TRF 1 binding protein, methods of use thereof

ABSTRACT

The present invention discloses a unique vertebrate protein, tankyrase that binds to telomeric repeat binding factor 1 (TRF1). Nucleic acids encoding tankyrases are also disclosed. Methods of screening drugs using tankyrase are also included.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present Application is a divisional of Ser. No. 09/196,387, filedNov. 19, 1998, now U.S. Pat. No. 6,277,613, which a Continuation-In-Partof U.S. Ser. No. 09/135,233 filed Aug. 17, 1998, now abandoned, which isa Continuation-In-Part of U.S. Ser. No. 09/095,225 filed Jun. 10, 1998,now abandoned the disclosures of which are hereby incorporated byreference in their entireties. Applicants claim the benefits of theseApplication under 35 U.S.C. § 120.

GOVERNMENTAL SUPPORT

The research leading to the present invention was supported, at least inpart, by a grant from the National Institutes of Health, Grant No. GM49046. Accordingly, the Government may have certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates generally to a unique vertebrate protein,tankyrase that binds to telomeric repeat binding factor 1 (TRF1), to thenucleic acids encoding tankyrases, and to therapeutic methods of usethereof. The tankyrases may also have a particular use in developingdrugs that can counteract the telomere shortening associated with agingand certain diseases such as ataxia telangiectasia.

BACKGROUND OF THE INVENTION

Telomeres are terminal structural elements found at the end ofchromosomes [Muller, The Collecting Net-Woods Hole, 13:181-195 (1939)]that protect natural double-stranded DNA ends from degradation, fusion,and recombination with chromosome-internal DNA [McClintock, Genetics,26:234-282 (1941); Lundblad et al., Cell, 87:369-375 (1996)]. Telomeresare also thought to play a role in the architecture of the nucleus[Agard et al., Nature, 302:676-681 (1983); Rabl, Morphol. J., 10:214-330(1885)], and to provide a solution to the end-replication problem thatarises as a consequence of successive replication of linear DNA by DNApolymerases which would otherwise result with progressively shorterterminal sequences [Watson, Nature, 239:197-201 (1972)]. In tetrahymena,impaired telomere function leads to a defect in cytokinesis and to celldeath [Yu et al., Nature, 344:126-132 (1990)]. Similarly, in yeast, lossof a single telomere results in cell cycle arrest and chromosomeinstability [Sandell and Zakian, Cell, 75:729-741 (1993)] and cellsundergoing generalized telomere shortening eventually senesce [Lundbladand Szostak, Cell, 57:633-643 (1989); Singer and Gottschling, Science,266:404-409 (1994)].

A ribonucleoprotein reverse transcriptase, telomerase, can elongatetelomeres using an internal RNA component as template for the additionof the appropriate G-rich sequence to the 3′ telomere termini [Greiderand Blackburn, Cell, 43:405-413 (1985)]. This activity is thought tocompensate for the inability of polymerases to replicate chromosomeends, but other mechanisms of telomere maintenance may operate as well[Pluta et al., Nature, 337:429-433 (1989)].

Telomeres contain a tandem array of repeat sequences, typically five toeight base pairs long, that are G-rich in the strand that extends to theend of the chromosome DNA. These repeat units appear to be bothnecessary and sufficient for telomere function [Lundblad and Szostak,Cell, 57:633-643 (1989); Szostak et al., Cell, 36:459-568 (1982)]. Alltelomeres of a single genome are composed of the same repeats and thesesequences are highly conserved across species. For instance, Oxytrichachromosomes terminate in TTTTGGGG repeats [Klobutcher et al., Proc.Natl. Acad. Sci. USA, 78:3015-3019 (1981)], Tetrahymena utilizes anarray of (TTGGGG)_(n) [Blackburn et al., J. Mol. Biol., 120:33-53(1978)], and plant chromosomes carry the sequence (TTTAGGG)_(n)[Richards et al., Cell, 53:127-136 (1988)]. Telomeres of trypanosomesand all vertebrates, including mammals, contain the repeat sequenceTTAGGG [Blackburn et al., Cell, 36:447-458 (1984); Brown, Nature,338:774-776 (1986); Cross et al., Nature, 338:771-774 (1989); Moyzis etal., Proc. Natl. Acad. Sci. USA, 85:6622-6626 (1988); Van der Ploeg etal., Cell, 36:459-468 (1984)]. This 6 basepair sequence is repeated inlong tandem arrays at the chromosome ends, which may be as long as 100kb in the mouse, and varies from 2 to 30 kb in humans [de Lange,Telomere Dynamics and Genome Instability in Human Cancer, In Telomeres,Blackburn and Greider eds., Cold Spring Harbor Press; 265-295 (1995)].

During the development of human somatic tissue, telomeres undergoprogressive shortening; in contrast, sperm telomeres increase with donorage [Broccoli et al., Proc. Natl. Acad. Sci. USA, 92:9082-9086 (1995);de Lange, Proc. Natl. Acad. Sci. USA, 91:2882-2885 (1994)]. Most if notall human somatic tissue chromosomes lose terminal TTAGGG repeats witheach division, e.g., about 15-40 basepairs per year in the skin andblood. It is unclear what effect this diminution has since humantelomeres are between 6-10 kb at birth. On the other hand, it is not yetknown how many kilobases of TTAGGG repeats are necessary for optimaltelomere function.

Primary human fibroblasts grown in culture lose about 50 basepairs oftelomeric DNA per doubling (PD) before they stop dividing at asenescence stage [Allsopp et al., Proc. Natl. Acad. Sci. USA,89:10114-10118 (1992)]. Importantly, there is an excellent correlationbetween the number of divisions that the cells go through and theirinitial telomere length. Indeed, it has been suggested that thecorrelation represents a molecular clock, which limits the potential ofprimary cells to replicate [Harley et al., Nature (London), 345:458-460(1990); Harley et al., Exp. Gerontol, 27:375-382 (1992)]. Thus,immortalization of human somatic cells involves a mechanism to halttelomere shortening [Bodnar et al., Science, 279:349-352 (1998)].

Changes in telomeric dynamics also appear to play a role in themalignant transformation of human cells [Counter et al., EMBO J.,11:1921-1929 (1992); Counter et al., Proc. Natl. Acad. Sci. USA,91:2900-2904 (1994); Kim et al., Science, 266:2011-2015 (1994)]. Forexample, telomeres of tumor cells are generally significantly shorterthan those of the corresponding normal cells [de Lange et al., Mol. CellBiol., 10:518-527 (1990)]. Telomerase activation appears to be anobligatory step in the immortalization of human cells [de Lange, Proc.Natl. Acad. Sci. USA, 91:2882-2885 (1994); Counter et al., EMBO J.,11:1921-1929 (1992); Counter et al., Proc. Natl. Acad. Sci.,91:2900-2904 (1994); Kim et al., Science, 266:2011-2015 (1994); Bodnaret al., Science, 279:349-352 (1998)].

Hanish et al. [Proc. Natl. Acad. Sci. USA, 91:8861-8865 (1994)] examinedthe requirements for the formation of human telomeres from TTAGGG seeds,and found that telomere formation was not correlated with the ability ofhuman telomerase to elongate telomeric sequences in vitro, and did notappear to be a result of homologous recombination. Rather, the sequencedependence of telomere formation matched the in vitro bindingrequirements for TRF1, a telomeric TTAGGG repeat binding protein that isassociated with human and mouse telomeres in interphase and in mitosis.

Indeed, several observations suggest the existence of regulatorymechanisms to control telomere length. Mammalian telomeres show aspecies-specific length setting [Kipling and Cooke, Nature, 347:400-402(1990)] indicating a mechanism to control telomere length in thegermline. Mammalian cells also have a mechanism to measure and regulatethe length of individual telomeres. For example, in telomere seedexperiments the final length of individual newly-formed telomeresmatches the length of the host cell telomeres [Barnett et al., Nucl.Acids Res., 21:27-36 (1993); Hanish et al., Proc. Natl. Acad. Sci. USA,91:8861-8865 (1994)]. Telomere length regulation is also apparent inseveral human cell lines, which maintain their telomeres at a stablelength setting despite high levels of telomerase [Counter et al., EMBOJ., 11: 1921-1929 (1992)]. Thus, cells can monitor and modulateindividual telomeres, a process that is likely to involve proteins boundto the TTAGGG repeats at chromosome ends.

Another process likely to be mediated by TTAGGG binding proteins is theprotective cap function of telomeres. Telomeres are protected from thecellular surveillance systems that monitor DNA damage. Thus, cells candistinguish natural chromosome ends (telomeres) from double strandbreaks (resulting from DNA damage).

The only known protein components of mammalian telomeres are the TRFproteins, duplex TTAGGG repeat binding factors that are localized attelomeres in interphase and metaphase chromosomes [Zhong et al., Mol.Cell. Biol., 13:4834-4943 (1992); Chong et al., Science, 270:1663-1667(1995); Ludérus et al., J. Cell Biol., 135:867-881 (1996); Broccoli etal., Hum. Mol. Genetics, 6:69-76 (1997); see Smith and de Lange, Trendsin Genetics, 13:21 -26 (1997) for review; Broccoli et al., Nature Gen.,17:231-235 (1997); Bilaud et al., Nature Gen., 17:236-239 (1997); vanSteensel et al., Cell, 92:401-413 (1998)]. Thus far, only two humantelomeric DNA binding proteins have been identified, TRF1 and TRF2 [U.S.Pat. No. 5,733,730, Issued Mar. 31, 1998, and U.S. patent applicationSer. No: 08/938,052, filed Sep. 26, 1997, and Ser. No. 09/018,636 filedFeb. 4, 1998, all of which are whereby incorporated by reference intheir entireties]. TRF1 was isolated as a double-stranded TTAGGG-repeatbinding protein from HeLa cells [Chong et al., Science, 270:1663-1667(1995)]. This factor contains three recognizable domains: an acidicN-terminal domain, a dimerization domain, and a C-terminal three helixbundle similar to the Myb and homeodomain DNA-binding folds [Bianchi etal., EMBO J., 16:1785-1794 (1997); Chong et al., Science, 270:1663-1667(1995); reviewed in Konig and Rhodes, Cell, 85:125-136 (1996); Smith andde Lange, Trends in Genetics, 13:21-26 (1997)]. A second factor, TRF2,is related to TRF1 in its dimerization domain and the C-terminal Mybmotif, but differs in that its N-terminus is basic rather than acidic[Bilaud et al., Nature Gen., 17:236-239 (1997); Broccoli et al., NatureGen., 17:231-235 (1997)]. Despite their related dimerization domains,the proteins do not interact with each other [Broccoli, et al., NatureGen., 17:231-235 (1997)], and probably exist predominantly ashomodimers. Both proteins bind specifically to double-stranded TTAGGGrepeats in vitro and are located at telomeres in vivo. The two TRFs areubiquitously expressed and current evidence indicates that most humantelomeres contain both factors bound simultaneously throughout the cellcycle [Broccoli et al., Nature Gen., 17:231-235 (1997); Chong et al.,Science, 270:1663-1667 (1995); Smith and de Lange, Trends in Genetics,13:21-26 (1997)]. Two other double-stranded telomeric-repeat bindingproteins have been identified; Rap1p in S. cerevisia [Reviewed in Shore,Trends Gen., 10:408-412 (1994) and Tazlp in S. pombe [Cooper et al.,Nature, 385:744-474 (1997)]. Both have Myb type DNA-binding domains[Cooper et al., Nature, 385:744-747 (1997); Konig et al., Cell,85:125-136 (1996)]. In addition, Tazlp shows weak overall homology withTFR1 and shares its acidic nature [Cooper et al., Nature, 385:744-747(1997)].

Recent studies have shown that TRF2 plays a key role in the protectiveactivity of telomeres by inhibiting end-to-end fusions [van Steensel etal., Cell, 92:401-413 (1998)]. Previous studies had indicated that TRF1plays a different role in telomere biology, functioning as a negativeregulator of telomere length maintenance [van Steensel and de Lange,Nature, 385:740-743 (1997)]. Thus, long-term overexpression of TRF1 in atelomerase-positive tumor cell line resulted in progressive telomereshortening. Conversely, removal of TRF1 from the telomere (throughexpression of a dominant negative mutant) induced telomere elongation.In these experiments TRF1 did not detectably alter the activity oftelomerase in cell extracts. Based on these observations it was proposedthat TRF1 negatively regulates telomerase at the level of individualtelomeres; an increase in the amount of TRF1 at the telomere wouldcreate a negative signal for telomerase, whereas, a decrease would senda positive signal to telomerase [van Steensel and de Lange, Nature,385:740-743 (1997)]. Interestingly, a similar mechanism of telomerelength regulation exists in yeasts where it has been shown that Taz1pand Rap1p function as negative regulators of telomere length. As is thecase for yeast telomere length regulation, the mechanism by which TRF1controls telomere synthesis by telomerase is not fully understood[Conrad et al., Cell, 63:739-750 (1990); Cooper et al., Nature,385:744-747 (1997); Lustig et al., Science, 250:549-553 (1990); Marcandet al., Science, 275:986-990 (1997); McEachern and Blackburn, Nature,376:403-409 (1995)].

Indeed, telomere homeostasis involves a balance of lengthening andshortening activities. The telomerase catalytic subunit produces thelengthening activity, whereas other proteins including the telomerebinding protein TRF1 are involved in establishing a telomere lengthequilibrium. Recently Bodnar et al. [Science 279:349-352 (1998)] haveshown that extremely low levels of telomerase activity are insufficientto prevent telomere shortening; a result that is consistent with theobservation that stem cells have low but detectable telomerase activity,yet continue to exhibit shortening of their telomeres throughout life.

Therefore, there is a need to isolate additional proteins, preferablyenzymes involved in telomere homeostasis. Furthermore, there is a needto characterize such proteins. In addition, there is a need to designand develop drug screens to identify agents that modulate such proteinsand thus can act as effectors on the important process of telomerelength homeostasis.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

SUMMARY OF THE INVENTION

The present invention provides an isolated and/or recombinant nucleicacid encoding a protein, tankyrase, that binds to TRF1. In anotherembodiment, the nucleic acid encodes a tankyrase-related protein. In oneembodiment the nucleic acid encodes a tankyrase or a tankyrase-relatedprotein that has an amino acid sequence that has at least 25% identitywith that of SEQ ID NO:2. In another embodiment the nucleic acid encodesa tankyrase or tankyrase-related protein comprising at least two,preferably three and more preferably all of the following domains: adomain that consists of homopolymeric tracts of histidine, proline andserine (HPS) preferably at the amino terminal end of the protein, anankyrin-specific (ANK) repeat consensus domain, a sterile alpha motif(SAM) motif, and a poly(ADP-ribose) polymerase (PARP)-related domain.Preferably the order of the domains is identical to that found in humantankyrase having the amino acid sequence of SEQ ID NO:2. The tankyraseis preferably an animal protein, more preferably a vertebrate protein,and even more preferably a mammalian protein. In the most preferredembodiment the tankyrase is a human protein. In one such embodiment theprotein is about 142-kDaltons and contains 24 ANK repeats, a SAM motif,a PARP-related domain, and an N-terminal domain rich in proline,histidine and serine (HPS). In another such embodiment tankyrase is aprotein that is relatively enriched in the nuclear envelope fraction andin a tight association with the nuclear envelope e.g., remaining boundto the nuclear envelope even after extraction with 0.5 M NaCl and 8 Murea. In a particular embodiment of this type the nucleic acid encodes atankyrase that is a human protein comprising the amino acid sequence ofSEQ ID NO:2. In a related embodiment of this type the nucleic acidencodes a tankyrase comprising the amino acid sequence of SEQ ID NO:2with a conservative amino acid substitution. In a more particularembodiment the nucleic acid comprises the coding sequence of SEQ IDNO:1. All of the recombinant and/or isolated nucleic acids of thepresent invention can further comprise a heterologous nucleotidesequence.

The present invention also provides nucleic acids, e.g., recombinant DNAmolecules that comprise a nucleotide sequence encoding a fragment of atankyrase that can bind to the acidic domain of a TRF1. In a preferredembodiment the fragment comprises at least a portion of the ANK repeatconsensus domain of the tankyrase. In a particular embodiment of thistype the nucleic acid encodes a fragment of the tankyrase that comprisesthe amino acids 436 to 796 of SEQ ID NO:2. In a related embodiment ofthis type the nucleic acid encodes a fragment of the tankyrase thatcomprises the amino acids 436 to 796 of SEQ ID NO:2 with a conservativeamino acid substitution. In another such embodiment the nucleic acidencodes a fragment of the tankyrase that comprises the amino acids 181to 1005 of SEQ ID NO:2. In a related embodiment of this type the nucleicacid encodes a fragment of the tankyrase that comprises the amino acids181 to 1005 of SEQ ID NO:2 with a conservative amino acid substitution.In still another embodiment of this type, the nucleic acid encodes afragment of the tankyrase that comprises the amino acids 336 to 1163 ofSEQ ID NO:2. In a related embodiment of this type the nucleic acidencodes a fragment of the tankyrase that comprises the amino acids 336to 1163 of SEQ ID NO:2 with a conservative amino acid substitution.

In another embodiment a nucleic acid, e.g., a recombinant DNA moleculecomprises a nucleotide sequence encoding a fragment of a tankyrasecomprising a PARP-related domain. In one such embodiment the nucleicacid comprises a nucleotide sequence encoding a fragment of a tankyrasecomprising the amino acids 1159 to 1314 of SEQ ID NO:2. In another suchembodiment the nucleic acid comprises a nucleotide sequence encoding afragment of a tankyrase comprising the amino acids 1159 to 1314 of SEQID NO:2 with a conservative amino acid substitution.

In still another embodiment a nucleic acid e.g., a recombinant DNAmolecule, comprises a nucleotide sequence encoding a fragment of atankyrase comprising a SAM motif. In one such embodiment the nucleicacid comprises a nucleotide sequence encoding a fragment of a tankyrasecomprising the amino acids 1023 to 1088 of SEQ ID NO:2. In another suchembodiment the nucleic acid comprises a nucleotide sequence encoding afragment of a tankyrase comprising the amino acids 1023 to 1088 of SEQID NO:2 with a conservative amino acid substitution. As is true for allof the nucleic acids of the present invention, all of the recombinantDNA molecules encoding fragments of a tankyrase can further comprise aheterologous nucleotide sequence.

In yet another embodiment, a nucleic acid, e.g., a recombinant DNAmolecule comprises a nucleotide sequence encoding a fragment oftankyrase comprising an HPS domain. In one such embodiment the nucleicacid comprises a nucleotide sequence encoding a fragment of a tankyrasecomprising the amino acids 1-180 of SEQ ID NO:2. In another suchembodiment the nucleic acid comprises a nucleotide sequence encoding afragment of a tankyrase comprising the amino acids 1-180 of SEQ ID NO:2with a conservative amino acid substitution.

The present invention also provides nucleic acids, e.g., recombinant DNAmolecules that comprise a nucleotide sequence encoding a truncatedtankyrase. In one such embodiment the nucleotide sequence comprises thecoding sequence for amino acid residues 1-640 of SEQ ID NO:2. In anotherembodiment, the nucleotide sequence comprises the coding sequence foramino acid residues 1-881 of SEQ ID NO:2. In one embodiment thenucleotide sequence encodes SEQ ID NO:8 or SEQ ID NO:8 with aconservative amino acid substitution. In a particular embodiment of thistype the nucleic acid has the nucleotide sequence of SEQ ID NO:7. Inanother embodiment the nucleotide sequence encodes SEQ ID NO:10 or SEQID NO:10 with a conservative amino acid substitution. In a particularembodiment of this type the nucleic acid has the nucleotide sequence ofSEQ ID NO:9.

Nucleic acids that hybridize to the nucleotide sequences that encode thetankyrases, fragments thereof including truncated tankyrases,tankyrase-related proteins, and fragments thereof are also included inthe present invention. In one such embodiment the nucleic acid is atleast about 24 nucleotides, preferably at least about 48 nucleotides,and more preferably at least about 96 nucleotides. In a preferredembodiment of this type, the nucleic acid encodes a tankyrase which hasat least one functional activity, preferably two and more preferablyall, of the activities of human tankyrase as disclosed herein. In aparticular embodiment the nucleic acid hybridizes to SEQ ID NO:1 undermoderately stringent conditions. In a preferred embodiment of this type,the nucleic acid hybridizes to SEQ ID NO:1 under high stringencyconditions.

The present invention further provides a nucleic acid that comprisesabout 15 or more, preferably about 24 or more, and more preferably about36 or more consecutive nucleotides from SEQ ID NO:1. In a preferredembodiment of this type, the nucleic acid encodes a tankyrase which hasat least one functional activity, preferably two, and more preferablyall of the functional activities of human tankyrase as disclosed herein.

In addition, the present invention also provides nucleotide probes forthe isolated and/or recombinant nucleic acids of the present invention.In a preferred embodiment of this type the nucleotide probe is for SEQID NO:1. Another nucleic acid that can be used as a probe contains thenucleotide sequence of SEQ ID NO:11. Still another nucleic acid that canbe used as a probe contains the nucleotide sequence of SEQ ID NO:12.

All of the nucleic acids of the present invention can be comprised by arecombinant DNA molecule that is operatively linked to an expressioncontrol sequence. The present invention further provides expressionvectors containing the recombinant DNA molecules of the presentinvention. In addition the present invention also provides methods ofexpressing a recombinant tankyrase protein or fragment thereof in a cellcontaining an expression vector of present invention. One suchembodiment comprises culturing the cell in an appropriate cell culturemedium under conditions that provide for expression of recombinanttankyrase or fragment thereof by the cell. Such methods can furtherinclude the step of purifying the recombinant tankyrase or fragmentthereof. The purified form of the recombinant tankyrases or fragmentsthereof are also included as part of the present invention. In onepreferred embodiment the nucleic acid encodes SEQ ID NO:2. In anotherpreferred embodiment, the nucleic acid encodes a fragment of thetankyrase that comprises the amino acids 436 to 796 of SEQ ID NO:2.

Another aspect of the present invention provides an isolated and/orrecombinant protein, tankyrase, that binds to TRF1. In anotherembodiment, the isolated and/or recombinant protein is atankyrase-related protein. In one embodiment the tankyrase ortankyrase-related protein has an amino acid sequence that has at least25% identity with that of SEQ ID NO:2. In another embodiment thetankyrase or tankyrase-related protein comprises at least two,preferably three, and more preferably all of the following domains: adomain rich in homopolymeric tracts of histidine, proline, and serine(HPS) which is preferably at the amino-terminal end of the protein, anankyrin-specific (ANK) repeat consensus domain, a sterile alpha motif(SAM) motif, and a poly(ADP-ribose) polymerase (PARP)-related domain.The tankyrase is preferably an animal protein, more preferably avertebrate protein, and even more preferably a mammalian protein. In themost preferred embodiment the tankyrase is a human protein. In one suchembodiment the protein is about 142-kDaltons and contains about 24 ANKrepeats, a SAM motif, an amino-terminus rich in histidine, proline andserine (i.e., an HPS domain), and a PARP-related domain. In another suchembodiment the tankyrase is a protein that is relatively enriched in thenuclear envelope fraction and in a tight association with the nuclearenvelope e.g., remaining bound to the nuclear envelope even afterextraction with 0.5 M NaCl and 8 M urea.

In another embodiment the present invention provides a tankyrase that isa human protein comprising the amino acid sequence of SEQ ID NO:2. In arelated embodiment of this type the tankyrase comprises the amino acidsequence of SEQ ID NO:2 with a conservative amino acid substitution. Thepresent invention further provides proteolytic fragments of thetankyrase proteins of the present invention. The present invention alsoprovides a protein comprising about 12 or more, preferably about 24 ormore, and more preferably about 36 or more consecutive amino acids fromSEQ ID NO:2 which functions as a tankyrase as disclosed herein.

The present invention also provides a fragment of a tankyrase that canbind to the acidic domain of a TRF1. In a preferred embodiment thefragment comprises at least a portion of the ANK repeat consensus domainof the tankyrase. In a particular embodiment of this type the fragmentof the tankyrase comprises the amino acids 436 to 796 of SEQ ID NO:2. Ina related embodiment of this type the fragment of the tankyrasecomprises the amino acids 436 to 796 of SEQ ID NO:2 with a conservativeamino acid substitution. In another such embodiment the fragment of thetankyrase comprises the amino acids 181 to 1005 of SEQ ID NO:2. In arelated embodiment of this type the fragment of the tankyrase comprisesthe amino acids 181 to 1005 of SEQ ID NO:2 with a conservative aminoacid substitution. In still another embodiment of this type, thefragment of the tankyrase comprises the amino acids 336 to 1163 of SEQID NO:2. In a related embodiment of this type the fragment of thetankyrase comprises the amino acids 336 to 1163 of SEQ ID NO:2 with aconservative amino acid substitution.

In still another embodiment a fragment of the tankyrase comprises an HPSdomain. In one such embodiment the fragment of a tankyrase comprises theamino acids 1 to 180 of SEQ ID NO:2. In another such embodiment thefragment of a tankyrase comprises the amino acids 1 to 180 of SEQ IDNO:2 with a conservative amino acid substitution.

In yet another embodiment a fragment of a tankyrase comprises aPARP-related domain. In one such embodiment the fragment of a tankyrasecomprises the amino acids 1159 to 1314 of SEQ ID NO:2. In another suchembodiment the fragment of a tankyrase comprises the amino acids 1159 to1314 of SEQ ID NO:2 with a conservative amino acid substitution.

In still another embodiment a fragment of a tankyrase comprises a SAMmotif. In one such embodiment the fragment of a tankyrase comprises theamino acids 1023 to 1088 of SEQ ID NO:2. In another such embodiment thefragment of a tankyrase comprises the amino acids 1023 to 1088 of SEQ IDNO:2 with a conservative amino acid substitution. All of the recombinantand/or isolated tankyrase proteins and fragments of the presentinvention can further be part of a chimeric and/or fusion peptide orprotein.

The present invention also provides truncated tankyrases. In one suchembodiment the truncated tankyrase comprises amino acid residues 1-640of SEQ ID NO:2. In another embodiment, the truncated tankyrase comprisesamino acid residues 1-881 of SEQ ID NO:2. In one embodiment thetruncated tankyrase comprises the amino acid sequence of SEQ ID NO:8 orSEQ ID NO:8 with a conservative amino acid substitution. In yet anotherembodiment the truncated tankyrase comprises the amino acid sequence ofSEQ ID NO:10 or SEQ ID NO:10 with a conservative amino acidsubstitution.

The present invention further provides antibodies to the proteins andfragments thereof including truncated proteins, and proteolyticfragments of the proteins of the present invention. In one suchembodiment the antibody is a polyclonal antibody. In another embodimentthe antibody is a monoclonal antibody. In still another embodiment theantibody is a chimeric antibody. The present invention further providesan immortal cell line that produces a monoclonal antibody of the presentinvention.

In another aspect of the present invention is a method of selecting acandidate drug that interferes with the binding of a tankyrase and aTRF1. One such embodiment comprises contacting a candidate drug with afirst protein or peptide comprising the acidic domain of a TRF1 and asecond protein or peptide comprising a tankyrase fragment that can bindto the acidic domain of a TRF1 under conditions where the first proteinor peptide and second protein or peptide bind in the absence of thecandidate drug and determining the binding between the first protein orpeptide and second protein or peptide; wherein a candidate drug isselected when the amount of binding determined in the presence of thedrug is measurably less than in its absence. Preferably the fragmentcomprises at least a portion of the ANK repeat consensus domain of thetankyrase.

The present invention further provides methods of selecting a candidatedrug that can modulate the PARP (and/or ARP) activity of a tankyrase.Such modulators can be either agonists or antagonists. Candidate drugsthat are selected as agonists cause an increase in PARP (or ARP)activity whereas candidate drugs that are selected as antagonists (e.g.,inhibitors) cause a decrease in PARP (and/or ARP) activity. One suchembodiment comprises contacting a candidate drug with a tankyrase or afragment of tankyrase that has PARP activity, NAD⁺ and a polyADP-ribosylating substrate under conditions in which the tankyrase (orthe fragment) polyADP-ribosylates the substrate in the absence of thecandidate drug. The polyADP-ribosylation state of the substrate (e.g., ahistone) is then determined. A candidate drug is selected as anantagonist when the polyADP-ribosylation state of the substratedetermined in the presence of the drug is measurably less than itsabsence. A candidate drug is selected as an agonist when thepolyADP-ribosylation state of the substrate determined in the presenceof the drug is measurably greater than its absence.

The present invention further provides methods of extending the lifespanof a non-tumor cell and/or inhibiting the growth of a tumor cell. Onesuch embodiment comprises administering an inhibitor to tankyrase. Inone particular embodiment of this type the inhibitor is3-aminobenzamide. In a preferred embodiment of this type the cell is ahuman cell.

Yet another aspect of the present invention comprises a method ofidentifying the sequence of a homologue to the human tankyrase gene. Onesuch embodiment comprises determining the homology of SEQ ID NO:2 to theamino acid sequences encoded by nucleic acids from a library of nucleicacids containing partial nucleotide sequences of coding regions ofgenes. Preferably this determination is aided by computer analysis. Anucleic acid containing a partial nucleotide sequence encoding a proteinthat is substantially homologous to SEQ ID NO:2 is then selected. Thesequence of the coding region of the gene is then determined. Thesequence is identified as being that of the homologue to the humantankyrase gene of the invention when it encodes a protein having anamino acid sequence that is substantially homologous to SEQ ID NO:2.

In one embodiment of the method, determining the sequence of the codingregion is performed by sequencing an insert of a plasmid which containsthe nucleic acid. In this case, the insert comprises the nucleic acid.In another embodiment, the method further comprises constructing arecombinant DNA that contains the coding region. In one such embodimenta recombinant protein is made by expressing the recombinant DNA. In apreferred embodiment of this type an activity of the tankyrase isassayed. In one such embodiment, the activity assayed is the ability ofthe recombinant protein to bind to TRF1. In another embodiment thesequence is identified as being that of the homologue to the humantankyrase gene when the recombinant protein has the activity of thehuman tankyrase. Recombinant DNA molecules and the recombinanttankyrases obtained by these methods are also part of the presentinvention.

The present invention further provides a method of transporting aprotein to the nucleus. This method arises from the identification ofthe mechanism by which tankyrase is carried into the nucleus by TRF1.More particularly, the present invention provides a nucleotide sequencethat encodes a protein or peptide of interest and a tankyrase fragmentthat can bind to the acidic domain of a TRF1. Minimally the fragment oftankyrase comprises at least a portion of the ANK repeat consensusdomain. A particularly useful aspect of this portion of the presentinvention is that the protein of interest can be localized to thetelomere. Such a protein can be used as a marker such as greenfluorescent protein, or for its particular activity such as a particularRNase, Dnase, or even a protein kinase. In a preferred embodiment ofthis type the nucleic acid encodes a fragment of the tankyrasecomprising the amino acids 436 to 796 of SEQ ID NO:2. In anotherembodiment of this type the nucleic acid encodes a fragment of thetankyrase comprising the amino acids 436 to 796 of SEQ ID NO:2 with aconservative amino acid substitution.

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show that the human tankyrase cDNA encodes a 142-kD proteincontaining 24 ANK repeats, a SAM motif and a PARP-related domain.

FIG. 1A shows the domain structure of tankyrase and TRF1. Lines belowthe schematic indicate inserts contained in the named plasmids used togenerate recombinant protein for antibody production. Numbers indicateamino acid residues in tankyrase.

FIG. 1B shows the predicted amino acid sequence of tankyrase. Analignment of the 24 ANK repeats is presented. Dashes within the repeatsindicate gaps and sequences to the right of the repeats indicateinsertions that occur after the underlined amino acid in the same line.Light shading indicates a match to the ANK repeat consensus derived byMichaely and Bennett [Trends Cell Biol., 2:127-129 (1992)] or by Bork[Proteins, 17:363-374 (1993)] and darker shading is a match to theankyrin-specific ANK repeat consensus derived from Peters and Lux [SeminHematol., 30:85-118 (1993)]. The SAM motif is doubly underlined and thePARP-related domain singly underlined.

FIG. 1C shows the amino acid sequence alignment of the tankyrase SAMmotif with Dm Bicaudal-C (Drosophila melanogaster Genbank #U15928), HsDiacyl (Homo sapiens Diacyl glycerol kinase delta, Genbank #D73409) andGg CEK9 (Gallus gallus chicken embryo kinase 9, Genbank # U23783).Identical residues found in HS tankyrase and at least one other sequenceare shaded. Numbers on the left indicate the amino acid residues in thecorresponding sequences.

FIG. 1D shows the amino acid sequence alignment of the PARP-relateddomain of tankyrase with Dm tankyrase (Drosophila Melanogaster EST,Genbank #AA391467), the catalytic domain of Hs PARP (Homo sapiens,Genbank # M32721) and DmPARP (Drosophila melanogaster Genbank # D13806)and a PARP-related domain in Hs KIAA0177 (Homo sapiens, Genbank#D79999). Identical residues found in Hs tankyrase and at least twosequences are shaded. Secondary structures are indicated by lineslabeled c, d, e, f, g, m, n (β-strands) and L (α-helix). Identical aminoacids conserved in the prokaryotic toxins, DT (diphtheria toxin) and ETA(exotoxin A), are indicated by an asterisk above the amino acid. Numberson the left indicate the amino acid residues in the correspondingsequences.

FIGS. 2A-2B show the expression of tankyrase mRNA and protein.

FIG. 2A is a Northern blot of polyadenylated RNAs derived form theindicated human tissues probed with a tankyrase cDNA. Asterisks indicatethe tankyrase transcripts. The blot was rehybridized with a β-actinprobe. PBL is peripheral blood leukocytes.

FIG. 2B is an immunoblot of proteins fractionated on 10% SDS-PAGE,transferred to nitrocellulose and probed with anti-tankyrase antibodies(lanes 1-3) or preimmune serum (lanes 4-6). Protein samples are: saltextracted nuclear pellet from rat testis (Testis) (lanes 1 and 4), wholecell lysates from HeLa cells (HeLa) (lanes 2 and 5) and products of acoupled in vitro transcription/translation reaction programmed with thetankyrase cDNA (IVTL) (lanes 3 and 6).

FIGS. 3A-3L show that the localization of exogenous tankyrase totelomeres is dependent upon TRF1. Hela 1 cells transfected withFLAG-tankyrase (FIGS. 3A-3D) or FLAG-tankyrase and TRF1 (FIGS. 3E-3L)were methanol-fixed and processed for indirect immunofluorescence. Cellswere double-stained with anti-FLAG antibody M2 (FIGS. 3A, 3E and 3I)(green) and anti-TRF1 antibody 371 (FIGS. 3B, 3F and 3J) (red). (FIGS.3C, 3G and 3K) indicates superimposition of the red and green images;yellow indicates colocalization of the red and green signal. DNA isstained with DAPI (D,H and L) (blue).

FIGS. 4A-4B show the analysis of tankyrase and TRF1 interaction byimmunoprecipitation and the two-hybrid assay.

FIG. 4A shows Cell extracts prepared from HeLa 1 cells transientlytransfected with TRF1 and FLAG-tankyrase-1 were subjected toimmunoprecipitation followed by immunoblot analysis. Proteins wereimmunoprecipitated with an unrelated rabbit serum as a control (C)(lanes 1 and 4), anti-tankyrase antibodies (tankyrase) (lanes 2 and 6)or anti-TRF1 antibody 371 (TRF1) (lanes 3 and 5). Samples wereprocessed, suspended in Laemmli buffer and divided in half. One set wasnot heated (left panel) and the other was heated at 100° C. for 5 min(right panel). Proteins were fractionated on 10% SDS-PAGE, transferredto nitrocellulose and probed with anti-TRF1 antibody 371 (left panel) oranti-tankyrase antibodies (right panel). Note that in the unheatedsample the IgGs are not fully reduced and migrate as an 80 kdaltonprotein. FIG. 4B shows the identification of the interacting domains ofTRF1 and tankyrase using the two-hybrid system. β-galactosidase levelswere measured for strains containing plasmids expressing LexA or variousLexA-TRF1 hybrids along with plasmids expressing the GAL4 activationdomain (GAD) or the GAD-tankyrase hybrid containing 10 internal ANKrepeats (9-19). The values represent an average of three independenttransformations. Values<0.01 are indicated by 0.

FIGS. 5A-5C show endogenous tankyrase localizes to nuclear porecomplexes.

FIG. 5A shows the co-localization of endogenous tankyrase with nuclearpore complex proteins by indirect immunofluorescence. Formaldehyde-fixedHela 1 cells were double-stained with anti-tankyrase antibodies (1) andMAb414 (2), a monoclonal antibody that recognizes a family of nuclearpore complex proteins.

FIG. 5B shows the immunblot analysis of co-fractionation of tankyrasewith nuclear envelopes. Subcelluar fractions of rat liver are: cytosol(C) (lane 1), crude nuclei (CN) (lane 2), nuclei (N) (lane 3),supernatant containing nuclear contents (S) (lane 4) and pelletcontaining nuclear envelopes (P) (lane 5) after DNAase digestion ofnuclei, supernatant (S) (lane 6) after extraction of nuclear envelopeswith 0.5 M NaCl, and supernatant (S) (lane 7) and pellet (P) (lane 8)after extraction of salt-washed nuclear envelopes with 8 M urea. Theamount of sample loaded for each fraction was based upon cellequivalents with an arbitrary value (x) for the starting number ofcells: 1x (lanes 1 and 2), 100x (lanes 3 and 4) and 1000x (lanes 5-8).Samples were either fractionated by 10% SDS-PAGE and proteins visualizedby staining with coomassie blue (top panel) or fractionated by 6.5%SDS-PAGE, transferred to nitrocellulose and probed with anti-tankyraseantibodies (bottom panel). Asterisks in the top panel indicate lamins A,B and C. Immunoreactive tankryin is indicated by an asterisk in thebottom panel.

FIG. 5C shows the localization of tankyrase to nuclear pore complexes byimmunoelectron microscopy. Formaldehyde-fixed Hela 1 cells were probedwith anti-tankyrase antibodies followed by 5 nm-gold-conjugatedanti-rabbit antibodies. Samples were processed by thin sectioningfollowed by analysis in the electron microscope. Shown are three panelsdepicting typical patterns of gold labeling of nuclear pore complexes.Magnification is 82,500 (top panel), 107,000 (bottom panels).

FIGS. 6A-6L show that the endogenous and exogenous tankyrase localizearound the pericentriolar matrix in mitotic cells. FIGS. 6A-6I are ofHeLa 1.2.11 cells that were methanol-fixed and double-stained withanti-tankyrase antibodies (FIGS. 6A, 6D, and 6G, green) and anti-centrinantibodies (FIG. 6B, red) or anti-γ-tubulin antibodies (FIG. 6E, red) oranti-NuMA antibodies (FIG. 6I, red). FIGS. 6J-6L show the indirectimmunofluorescence analysis of exogenous tankyrase. Hela 1 cells weretransfected with FLAG-tankyrase, methanol-fixed and double-stained withanti-FLAG antibody M2 (FIG. 6J, green) and anti-γ-tubulin antibodies(FIG. 6K, red). (Merge) FIGS. 6C, 6F, 6I and 6L indicate superimpositionof the red and green images; yellow indicates co-localization of the redand green signal. DAPI staining of DNA is shown in blue.

FIGS. 7A-7F show that endogenous tankyrase colocalizes with TRF1 totelomeres. Indirect immunofluorescence analysis of methanol-fixed HeLa1.2.11 cells (FIGS. 7A-7C) or swollen, formaldehyde-fixed metaphasespreads from Hela 1.2.11 cells (FIGS. 7D-7F) by double-staining withanti-tankyrase antibodies (FIGS. 7A and 7D, green) and anti-TRF1antibody #2 (FIGS. 7B and 7E, red). (Merge) FIGS. 7C and 7F indicatessuperimposition of the red and green images and yellow indicatescolocalization of the red and green signal. DAPI staining of DNA isshown in blue.

FIGS. 8A-8B show the role of human tankyrase in meiosis and telomerelength regulation.

FIG. 8A shows that tankyrase plays a role in the assembly of the bouquetstructure during prophase of meiosis I. Tankyrase mediates attachment oftelomeres to the nuclear envelope and the subsequent clustering oftelomeres at the centrosome.

FIG. 8B shows that TRF1 recruits tankyrase to long telomeres to inhibittelomerase. Long telomeres or an increase in TRF1 expression induces ahigher order structure that promotes tankyrase binding. TankyraseADP-ribosylates telomerase rendering it inactive.

FIGS. 9A-9E demonstrates that tankyrase is a poly(ADP-ribose) polymerasethat inhibits TRF1 in vitro.

In FIG. 9A Tankyrase is shown to ADP-ribosylates itself and TRF1.Tankyrase was allowed to modify itself and TRF1 in the presence of[³²P]NAD⁺ and the products were analyzed by Coomassie-Blue staining(left) and autoradiography (right) of SDS-PAGE gels (see Example 1,below). Reactions contained the proteins indicated above the lanes atthe following amounts: TRF1 at 4 μg (+), tankyrase at 4 μg (+) or at arange of 0, 0.8, and 4 μg (triangle). All reactions contained 1.3 μM[³²P]NAD⁺ (+) and three reactions were also supplemented with increasingamounts of cold NAD⁺ (0.04, 0.2, and 1 mM, triangle).

FIG. 9B demonstrates that ADP-ribosylation activity is intrinsic fortankyrase. Tankyrase was immunoprecipitated with preimmune orα-tankyrase antibodies as indicated and incubated in a PARP assay with[³²P]NAD⁺ and the products were detected by autoradiography (see Example1, below).

FIG. 9C shows that Tankyrase is inhibited by the PARP inhibitor3-aminobenzamide (3AB). Reactions containing 4 μg tankyrase (+), without(−) or with (+) 4 μg TRF 1, and 1.3 μM [³²P]NAD⁺ were incubated without(−) or with (+) 1 mM 3AB and processed as in FIG. 9A.

FIG. 9D demonstrates that Tankyrase products contain poly(ADP-ribose).Tankyrase and TRF1 were added as in panel FIG. 9C. Reactions for theleft panel contained no NAD⁺ (−) or 1.3 μM [³²P]NAD⁺ supplemented with 1μM or 1 mM cold NAD⁺ (triangle). Reactions for the right panel wereidentical to the reactions on the left but lacked labeled NAD⁺. Productswere transferred to nitrocellulose and autoradiographed (left) orimmunoblotted with monoclonal antibody 10H to poly(ADP-ribose) (rightpanel) (see Example 1, below).

FIG. 9E shows the inhibition of TRF-1 by Tankyrase. The Gel-shift assayfor the TTAGGG repeat binding activity of TRF1 used a duplex [TTAGGG]₁₂DNA as a probe. Binding reactions contained the components indicatedabove the lanes. Tankyrase was varied from 2.5 to 200 ng per 20 μlincubation in three-fold dilution steps (triangles). TRF1 was eitherpresent at 13 ng (+) or varied from 120 to 13 ng in three-fold dilutionsteps (triangle). NAD⁺ was at 0 (−) or 0.2 mM (+). The asterisksindicate the position of TRF1-containing complexes as determined byantibody super-shift experiments.

DETAILED DESCRIPTION OF THE INVENTION

A novel telomeric protein tankyrase (TRF1 interacting ankyrin) has beenidentified by a two-hybrid screen with TRF1. Tankyrase is the thirdmammalian telomeric protein to be described and it differs in severalrespects from the previously identified factors TRF1 and TRF2. Forexample, the predicted amino acid sequence of tankyrase indicates anovel domain organization, completely unrelated to TRF1 and TRF2. Inaddition, tankyrase localizes to telomeres not via the binding oftelomeric DNA like TRF1 and TRF2, but rather, through protein-proteininteraction with TRF1. A human tankyrase, as exemplified below, carriesa region of 24 ankyrin repeats (a hallmark of the ankyrin family) thatincludes the TRF1 binding site, a sterile alpha motif (SAM) proteininteraction motif, and a C-terminal domain with significant homology tothe catalytic domain of poly(ADP-ribose) polymerase (PARP), an enzymeinvolved in DNA repair and genome stability. Tankyrase binds to thetelomeric protein TRF1, which is a negative regulator of telomere lengthmaintenance.

Expression of the tankyrase cDNA in HeLa cells revealed a telomericstaining pattern, but only when tankyrase was co-transfected with TRF1,indicating a possible link between tankyrase localization to telomeresand TRF1 synthesis. Analysis of the subcellular distribution of theendogenous protein indicated that tankyrase co-localized with TFR1 totelomeres in interphase and mitosis. In addition to its telomericlocation, tankyrase is located at nuclear pore complexes duringinterphase and at centrosomes in mitosis.

Given its strong homology to PARP, tankyrase not surprisingly functionsas an enzyme and as such represents the first indication for anenzymatic activity other than telomerase associated with eukaryotictelomeres. Indeed, the PARP-related domain of tankyrase appears to beinvolved in the telomere length regulation by TRF1, and could directlymodify the effect of TRF1 on telomeres.

It has only recently become apparent that telomere dynamics plays amajor role in the life-cycle of a cell. The regulation of telomerelength has been implicated in the process of aging, as well as incancer, and other human diseases. For example, the mutation in ataxiatelangiectasia has recently be shown to confer a predisposition toaccelerated telomere shortening in peripheral blood lymphocytes[Metcalfe et al., Nature Genetics, 13:350-353 (1996)].

Telomeres undergo progressive shortening during the development of humansomatic tissue. Such telomere shortening eventually limits cellproliferation and leads to aging. Consistently, the number of celldivisions that primary human fibroblasts go through in culture isdependent on their initial telomere length. This correlation correspondsto a molecular clock that limits the potential of primary cells toreplicate, and indicates that immortalization of human somatic cellsinvolves a mechanism that must halt normal telomere shortening. Thisimplies that successfully inducing the elongation of telomeres, eitherin vitro or in vivo, could counteract this aspect of the aging process,and furthermore, could extend the life-span of human cells and tissues.In this capacity tankyrase, by inhibiting the action of TRF1 could actas such a counteracting agent.

On the other hand cancer cells appear to have the ability to maintaintheir telomeres at specific lengths. Not surprisingly, many cancer cellscontain the enzyme telomerase, which acts to lengthen telomeres andthereby counteract the shortening of the telomere that would otherwiseoccur during normal cellular division. Major efforts in thepharmaceutical industry are currently focused on telomerase as a targetin cancer chemotherapy. The rationale of this approach is thatinhibition of telomerase should lead to telomere shortening in thetumors and this process is eventually expected to halt proliferation ofthe cancer cells. In addition, telomeres of cancer cells are generallysignificantly shorter than those of the corresponding normal cells. Thisdecrease in telomere length may be a factor in the instability of thegenome of cancer cells. Since there is evidence that tankyrase can actas a regulatory enzyme the inhibition of tankyrase could potentiallylead to consequences that are detrimental to the cell including tumorcells [McEachern and Blackburn, Nature, 376:403 -409 (1995)].

In humans, telomere maintenance is controlled by a negative feedbackmechanism that stabilizes telomeres in telomerase-expressing cells. TRF1plays a role in the regulation of telomere length. TRF1 performs itsrole in the regulation, at least in part, by binding to telomeres andinhibiting telomerase-catalyzed telomere elongation. As disclosedherein, TRF1 may also regulate telomere length by being a bindingpartner to tankyrase. Long term overexpression of TRF1s intelomerase-positive tumor cell lines results in a gradual andprogressive telomere shortening, whereas the expression of adominant-negative allele encoding an A-TRF, a specific inhibitor ofTRF1, inhibits binding of endogenous TRF1 to telomeres, and therebypermits telomere elongation. Importantly, the affinity of tankyrase forTRF1 appears to increase when TRF1 is bound to the telomere.

Inhibition of TRF1 binding to telomeres has been shown to lead totelomere elongation of cells expressing telomerase in vitro. Based onthis data it follows that in vivo inhibition of TRF1 will result intelomere elongation in cells that express telomerase. Telomerase isexpressed in self-renewing tissues such as bone marrow cells, peripheralblood T and B cells, and in basal keratinocytes. In these cells, and inother normal human cells that express telomerase, inhibition of TRF1 dueto tankyrase activity should lead to telomere elongation and concomitantextension of life-span.

Aside from the myriad of therapeutic applications for cells containingrecombinant tankyrase of the present invention, cells having an extendedlife-span due, at least in part to the presence of modulators oftankyrase activity, obtained by the methods described herein, can alsohave important ex vivo applications such as in the production ofbioengineered products. Indeed, cells in which telomere length can bemanipulated are an important tool for basic analysis of telomerestructure, function, and dynamics and for the analysis of telomerasefunction and regulation. In addition, the manipulation of telomerelength provides insight in the relationship between telomere dynamicsand cellular life-span. The disclosure of a new protein involved intelomere homeostasis provides new avenues for drug design and geneticmanipulations.

More specifically, the direct correlation shown between telomeremaintenance and cellular senescence, by [Bodner et al., Science,279:349-352 (1993)] for example indicates that the compositions andprocesses provided by the present invention can also play a direct rolein preventing and/or treating (1) atrophy of the skin through loss ofextracellular matrix homeostasis in dermal fibroblasts [Takeda et al.,Arch. Dermatol. 130:87 (1994)]; (2) age-related macular degeneration[Bouton et al., J. Neurosci. 15:4992 (1995)]; (3) and atherosclerosis[Kumazaki et al., J. Med. Sci., 42:97 (1993)]. In addition, Bodner etal., [supra] has pointed out that cells having an extended life-span canalso have important ex vivo applications in the production ofbioengineered products such as recombinant proteins. Furthermore, theextension of cellular in vitro life-span of normal human cells throughthe teachings of the present invention also has applications in creatinglarge populations of normal human cells in the laboratory. Large numbersof human cells can be important for generation of tissues (e.g. skin forburn victims) and even stem cells, including for creation of cellpopulations used in ex vivo gene-therapy.

Therefore, if appearing herein, the following terms shall have thedefinitions set out below:

As used herein the term “tankyrase” is used interchangeably with theterm “tankyrin” and refers to a protein that has binding affinity forTRF1, and more specifically the N-terminal acidic domain of a TRF1.Tankyrase contains an ankyrin-specific (ANK) repeat consensus domain, asterile alpha motif (SAM) motif; and a poly(ADP-ribose) polymerase(PARP)-related domain as described below. Preferably it also contains adomain that is rich in proline, histidine and serine (HPS), and morepreferably the HPS domain is at the amino terminal end of the protein.Tankyrase also has PARP activity in vitro with either TRF1 and/ortankyrase functioning as acceptors/substrates for ADP-ribosylation. Asexemplified below a human tankyrase has the amino acid sequence of SEQID NO:2 which is encoded by the nucleotide sequence of SEQ ID NO:1. Atankyrase is a specific type of tankyrase-related protein defined below.

As used herein, a “tankyrase-related protein” is a protein that isidentified as a member of a family of proteins by its structuralsimilarity with human tankyrase as exemplified herein by the firstmember, human tankyrase having SEQ ID NO:2. Minimally a“tankyrase-related protein” contains at least two structural and/orfunctional domains in common with human tankyrase, (e.g., an HPS, ANK,SAM or PARP-like domain), or alternatively has about at least 25% aminoacid identity with human tankyrase having an amino acid sequence of SEQID NO:2 over a contiguous block of about 1300 amino acid residues,preferably taking into account any particular deletions or insertionsthat could otherwise alter the correspondence between the two amino acidsequences, or alternatively has about at least 25% amino acid homologywith human tankyrase having an amino acid sequence of SEQ ID NO:2 asdetermined as a percent likeness of the amino acid sequence of thetankyrase-related protein with SEQ ID NO:2 with a standard computeranalysis that is comparable, and preferably identical to that determinedwith an Advanced Blast search at www.ncbi.nlm.nih.gov under the defaultfilter conditions. In a preferred embodiment the “tankyrase-relatedprotein” has two or more of these properties. In addition, it ispreferable that the protein has at least three domains in common with ahuman tankyrase and more preferably it has all four of these domains. Inthe most preferred embodiment of this type, the order of the domains isidentical to that of human tankyrase as disclosed herein. Similarly, itis preferable that the tankyrase-related protein has about at least 50%amino acid identity, and more preferably at least about 75%, and evenmore preferably at least about 90% amino acid identity with humantankyrase having the amino acid sequence SEQ ID NO:2 over a contiguousblock of about 1300 amino acid residues. Alternatively, it is alsopreferable that the tankyrase-related protein have about at least 50%amino acid homology and more preferably at least about 75% and even morepreferably at least about 90% amino acid homology with human tankyrasehaving the amino acid sequence SEQ ID NO:2, with the homology determinedby a standard computer analysis as cited above. A tankyrase-relatedprotein preferably functions in at least one respect like humantankyrase, e.g., binding a TRF such as TRF1, having an ADP-ribosylatingactivity, and/or having a role in telomere function. More preferably thetankyrase-related protein binds a TRF such as TRF1, has anADP-ribosylating activity, and has a role in telomere function.

As used herein, an “ANK” domain is a protein domain that contains 24ankyrin-specific repeats [Bork, Proteins, 17:363-374 (1993); Michaelyand Bennett, Trends Cell Biol., 2:127-129 (1992) and Bennett, J. Biol.Chem., 267:8703-8706 (1992)].

As used herein, a “SAM” domain is a sterile alpha motif, i.e., a 65-70amino acid domain found in 1-3 copies in a diverse group of proteinsimplicated in developmental processes [Ponting, Protein Science,4:1928-1930 (1995); Schultz et al., Protein Science, 6:249-253 (1997)].

As used herein, a “PARP-like” domain is a protein domain thatcorresponds to the PARP domain as described by Domenighini et al. [Mol.Microbiol., 14:41-50 (1994) and Ruf et al., Proc. Natl. Acad. Sci. USA,93:7481-7485 (1996)].

As used herein, an “HPS” domain is a protein domain contained within aregion of about 150 consecutive amino acids or less, in whichhomopolymeric runs (or tracts) of histidine, proline, and serine arefound. The polymeric runs minimally contain 5 consecutive prolines, or 5consecutive histidines, or 5 consecutive serines. An HPS domain isexemplified in the N-terminal region of the amino acid sequence of SEQID NO:2.

The telomeric repeat binding factor 1, TRF1, plays a role in theregulation of telomere maintenance by acting as a negative regulator oftelomere elongation is a dimeric protein that binds to a specifictelomeric repeat sequence found at the ends of telomeres [U.S. Pat. No.5,733,730, Issued Mar. 31, 1998, and U.S. patent application Ser. No.:08/938,052, filed Sep. 26, 1997, and Ser. No. 09/018,636 filed Feb. 4,1998, all of which are hereby incorporated by reference in theirentireties]. In vertebrates, the telomeric repeat sequence is TTAGGG.TRF1 has three distinct structural domains, a DNA binding domainencompassing the region of the protein that binds to the specifictelomeric repeat sequence, a dimerization domain encompassing the regionof the monomer that binds to its geminate partner to form a dimer, andan N-terminal acidic region that binds to tankyrase as described below.

The term “telomeric repeat binding factor 2”, or “TRF2”, is a telomericprotein that is required to maintain the correct structure at telomeretermini, and thereby protect against end-to-end fusions [U.S. patentapplication Ser. No: 08/938,052, filed Sep. 26, 1997; and Ser. No.09/018,636 filed Feb. 4, 1998]. TRF2, therefore, plays a role in thesuccessful progression through the cell division cycle. As such, TRF2 isinvolved in the main functions ascribed to telomeres in somatic humancells and is therefore a player in the loss of telomere function andgrowth arrest that accompanies telomere shortening in normal andtransformed human cells. TRF2 has three distinct structural domains, aDNA binding domain encompassing the region of the protein that binds tothe specific telomeric repeat sequence, a dimerization domainencompassing the region of the monomer that binds to its geminatepartner to form a dimer, and an N-terminal basic region.

As used herein an “altered TRF” (“A-TRF”) is a modified vertebrate TRFthat binds to TRF to form a heterodimer [U.S. patent application Ser.No. 08/800,264, filed Feb. 14, 1997, and Ser. No. 09/018,628 filed Feb.4, 1998 hereby incorporated by reference in their entireties.]. Theresulting heterodimer has a measurably lower binding affinity for theTRF telomeric repeat sequence than does the corresponding TRF homodimer.Thus the A-TRF hinders and/or prevents the binding of the correspondingTRF to its telomere repeat sequence binding site. An “A-TRF1” is analtered TRF1, whereas an “A-TRF2” is an altered TRF2.

As used herein the terms “approximately” and “about” are used to signifythat a value is within ten percent of the indicated value i.e., aprotein fragment containing “approximately” 140 amino acid residues cancontain between 126 and 154 amino acid residues.

As used herein the term “binds to” is meant to include all such specificinteractions that result in two or more molecules showing a preferencefor one another relative to some third molecule. This includes processessuch as covalent, ionic, hydrophobic and hydrogen bonding but does notinclude non-specific associations such solvent preferences.

As used herein, the term “homologue” refers to the relationship betweenproteins that have a common evolutionary origin and differ because theyoriginate from different species. For example, chicken TRF2 is ahomologue of human TRF2.

Genes Encoding Tankyrase

The present invention contemplates isolation of a gene encoding atankyrase or a tankyrase-related protein, preferably from a vertebrate,including a full length, or naturally occurring form of tankyrase fromany species, preferably an animal, more particularly mammalian, and evenmore particularly a human source.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed.1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

As used herein, the term “gene” refers to an assembly of nucleotidesthat encode a polypeptide, and includes cDNA and genomic DNA nucleicacids.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” is any genetic element (e.g.,plasmid, chromosome, virus) that functions as an autonomous unit of DNAreplication in vivo, i.e., capable of replication under its own control.

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been “transformed”by exogenous or heterologous DNA when the transfected DNA effects aphenotypic change. Preferably, the transforming DNA should be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

A “heterologous nucleotide sequence” as used herein is a nucleotidesequence that is added to a nucleotide sequence of the present inventionby recombinant methods to form a nucleic acid which is not naturallyformed in nature. Such nucleic acids can encode chimeric and/or fusionproteins. Thus the heterologous nucleotide sequence can encode peptidesand/or proteins which contain regulatory and/or structural properties.In another such embodiment the heterologous nucleotide can encode aprotein or peptide that functions as a means of detecting the protein orpeptide encoded by the nucleotide sequence of the present inventionafter the recombinant nucleic acid is expressed. In still another suchembodiment the heterologous nucleotide can function as a means ofdetecting a nucleotide sequence of the present invention. A heterologousnucleotide sequence can comprise non-coding sequences includingrestriction sites, regulatory sites, promoters and the like.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA—RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). However, unless specifically stated otherwise,a designation of a nucleic acid includes both the non-transcribed strandreferred to above, and its corresponding complementary strand. Suchdesignations include SEQ ID NOs:. A “recombinant DNA molecule” is a DNAmolecule that has undergone a molecular biological manipulation.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m) of55°, can be used, e.g., 5× SSC, 0.1% SDS, 0.25% milk, and no formamide;or 30% formamide, 5× SSC, 0.5% SDS). Moderate stringency hybridizationconditions correspond to a higher T_(m), e.g., 40% formamide, with 5× or6× SCC. High stringency hybridization conditions correspond to thehighest T_(m), e.g., 50% formamide, 5× or 6× SCC. Hybridization requiresthat the two nucleic acids contain complementary sequences, althoughdepending on the stringency of the hybridization, mismatches betweenbases are possible. The appropriate stringency for hybridizing nucleicacids depends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of T_(m) for hybrids of nucleic acids having those sequences.The relative stability (corresponding to higher T_(m)) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating T_(m) have been derived (see Sambrook et al.,supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7 -11.8). Preferably a minimum length for ahybridizable nucleic acid is at least about 12 nucleotides; preferablyat least about 16 nucleotides; and more preferably the length is atleast about 24 nucleotides; and most preferably 36 nucleotides.

In a specific embodiment, the term “standard hybridization conditions”refers to a T_(m) of 55° C., and utilizes conditions as set forth above.In a preferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 65° C.

“Homologous recombination” refers to the insertion of a foreign DNAsequence of a vector in a chromosome. Preferably, the vector targets aspecific chromosomal site for homologous recombination. For specifichomologous recombination, the vector will contain sufficiently longregions of homology to sequences of the chromosome to allowcomplementary binding and incorporation of the vector into thechromosome. Longer regions of homology, and greater degrees of sequencesimilarity, may increase the efficiency of homologous recombination.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be expressed on the surface of a cell. This sequenceencodes a signal peptide, N-terminal to the mature polypeptide, thatdirects the host cell to translocate the polypeptide. The term“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms.

As used herein, the term “sequence homology” in all its grammaticalforms refers to the relationship between proteins that possess a “commonevolutionary origin, ” including proteins from superfamilies (e.g., theimmunoglobulin superfamily) and homologous proteins from differentspecies (e.g., myosin light chain, etc.) [Reeck et al., Cell, 50:667(1987)].

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that do not share a commonevolutionary origin [see Reeck et al., 1987, supra]. However, in commonusage and in the instant application, the term “homologous,” whenmodified with an adverb such as “highly,” may refer to sequencesimilarity and not a common evolutionary origin.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

Similarly, in a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when greater than30% of the amino acids are identical, or greater than about 60% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program, using the default parameters.

The term “corresponding to” is used herein to refer similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. Thus, the term “corresponding to” refers to the sequencesimilarity over a given sequence range (e.g. 50 nucleotides), and notthe numbering of the amino acid residues or nucleotide bases.

A gene encoding a tankyrase or a tankyrase-related protein, whethergenomic DNA or cDNA, can be isolated from any source, particularly froma human cDNA or genomic library. Methods for obtaining a tankyrase genewith the nucleotide information disclosed herein is well known in theart [see, e.g., Sambrook et al., 1989, supra].

Accordingly, any animal cell potentially can serve as the nucleic acidsource for the molecular cloning of a tankyrase gene. The DNA may beobtained by standard procedures known in the art from cloned DNA (e.g.,a DNA “library”), by chemical synthesis, by cDNA cloning, or by thecloning of genomic DNA, or fragments thereof, purified from the desiredcell [see, for example, Sambrook et al., 1989, supra; Glover, D. M.(ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford,U.K. Vol. I, II]. Clones derived from genomic DNA may contain regulatoryand intron DNA regions in addition to coding regions; clones derivedfrom cDNA will not contain intron sequences. Whatever the source, thegene should be molecularly cloned into a suitable vector for propagationof the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired tankyrase gene may be accomplished in anumber of ways. For example, if an amount of a portion of a tankyrasegene or its specific RNA, or a fragment thereof, is available and can bepurified and labeled, the generated DNA fragments may be screened bynucleic acid hybridization to the labeled probe [Benton and Davis,Science, 196:180 (1977); Grunstein and Hogness, Proc. Natl. Acad. Sci.U.S.A., 72:3961 (1975)]. For example, a set of oligonucleotidescorresponding to the partial amino acid sequence information obtainedfor the tankyrase protein can be prepared and used as probes for DNAencoding a tankyrase. Preferably, a fragment is selected that is highlyunique to a tankyrase. Those DNA fragments with substantial homology tothe probe will hybridize. As noted above, the greater the degree ofhomology, the more stringent hybridization conditions can be used. In aspecific embodiment, stringent hybridization conditions are used toidentify a homologous tankyrase gene.

Further selection can be carried out on the basis of the properties ofthe gene, e.g., if the gene encodes a protein product having theisoelectric, electrophoretic, amino acid composition, or partial aminoacid sequence of a tankyrase as disclosed herein. Thus, the presence ofthe gene may be detected by assays based on the physical, chemical, orimmunological properties of its expressed product. For example, cDNAclones, or DNA clones which hybrid-select the proper mRNAs, can beselected which produce a protein that, e.g., has similar or identicalelectrophoretic migration, isoelectric focusing or non-equilibrium pHgel electrophoresis behavior, proteolytic digestion maps, or antigenicproperties as known for a tankyrase.

A tankyrase or tankyrase-related protein gene can also be identified bymRNA selection, i.e., by nucleic acid hybridization followed by in vitrotranslation. In this procedure, nucleotide fragments are used to isolatecomplementary mRNAs by hybridization. Such DNA fragments may representavailable, purified tankyrase DNA, or may be synthetic oligonucleotidesdesigned from the partial amino acid sequence information.Immunoprecipitation analysis or functional assays (e.g., tankyraseactivity) of the in vitro translation products of the products of theisolated mRNAs identifies the mRNA and, therefore, the complementary DNAfragments, that contain the desired sequences. In addition, specificmRNAs may be selected by adsorption of polysomes isolated from cells toimmobilized antibodies specifically directed against tankyrase.

The nucleotide sequence of the human tankyrase, SEQ ID NO:1 can also beused to search for highly homologous genes from other species, or forproteins having at least one homologous domain, using computer databases containing either partial or full length nucleic acid sequences.Human ESTs, for example, can be searched. The human tankyrase sequencecan be compared with human sequences, e.g., in GenBank, using GCGsoftware and the blast search program for example. Matches with highlyhomologous sequences or portions thereof can then be obtained.

If the sequence identified is an EST, the insert containing the EST canbe obtained and then fully sequenced. The resulting sequence can then beused in place of, and/or in conjunction with SEQ ID NO:1 to identifyother ESTs which contain coding regions of the tankyrase homologue (ortankyrase domain homologue). Plasmids containing the matched EST forexample can be digested with restriction enzymes in order to release thecDNA inserts. If the plasmid does not contain the full length homologuethe digests can be purified, e.g., run on an agarose gel and the bandscorresponding to the inserts can be cut from the gel and purified. Suchpurified inserts are likely to contain overlapping regions which can becombined as templates of a PCR reaction using primers which arepreferably located outside of the tankyrase open reading frame.Amplification should yield the expected product which can be ligatedinto a vector and used to transform an E coli derivative e.g., via TAcloning (Invitrogen) for example. A resulting full-length tankyrasehomologue can be placed into an expression vector and the expressedrecombinant tankyrase can then be assayed for TRF1 binding activity.

Alternatively, plasmids containing matched EST homologue fragments canbe used to transform competent bacteria (e.g, from Gibco BRL,Gaithersburg Md.). Bacteria can be streaked, then grown up overnight.Plasmid preps can be performed (e.g., Quiagen Corp, Santa ClaritaCalif.) and the plasmids can be digested by simultaneous restrictiondigest. Products of the digest can be separated by size on an agarosegel, for example, and purified. The corresponding bands cut from thesegels can be ligated to form a full length tankyrase cDNA and used totransform competent bacteria and the resulting plasmid can be purified.

A radiolabeled tankyrase cDNA can be synthesized using the selected mRNA(from the adsorbed polysomes) as a template. The radiolabeled mRNA orcDNA may then be used as a probe to identify homologous tankyrase DNAfragments from among other genomic DNA fragments.

The present invention also relates to cloning vectors containing genesencoding the domains of the tankyrases of the invention. The productionand use of such derivatives and analogs are within the scope of thepresent invention.

A modified tankyrase can be made by altering nucleic acid sequencesencoding the tankyrase by making substitutions, additions or deletionsthat provide for functionally equivalent molecules. Preferably, suchderivatives are made that have enhanced or increased effect on telomereelongation relative to the tankyrase. For example, a preferred tankyrasemay bind TRF1 more tightly than the native form.

Due to the degeneracy of nucleotide coding sequences, other DNAsequences which encode substantially the same amino acid sequence as atankyrase gene may be used in the practice of the present inventionincluding those comprising conservative substitutions thereof. Theseinclude but are not limited to modified allelic genes, modifiedhomologous genes from other species, and nucleotide sequences comprisingall or portions of tankyrase genes which are altered by the substitutionof different codons that encode the same amino acid residue within thesequence, thus producing a silent change. Likewise, the tankyrasederivative of the invention can include, but is not limited to, thosecontaining, as a primary amino acid sequence, all or part of the aminoacid sequence of a tankyrase protein including altered sequences inwhich functionally equivalent amino acid residues are substituted forresidues within the sequence resulting in a conservative amino acidsubstitution. And thus, such substitutions are defined as a conservativesubstitution.

For example, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity, which acts as afunctional equivalent, resulting in a silent alteration. Substitutes foran amino acid within the sequence may be selected from other members ofthe class to which the amino acid belongs. For example, the nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. Amino acidscontaining aromatic ring structures are phenylalanine, tryptophan, andtyrosine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such alterations will not be expected to significantlyaffect apparent molecular weight as determined by polyacrylamide gelelectrophoresis, or isoelectric point.

Particularly preferred conservative substitutions are:

Lys for Arg and vice versa such that a positive charge may bemaintained;

Glu for Asp and vice versa such that a negative charge may bemaintained;

Ser for Thr such that a free —OH can be maintained; and

Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced at a potential site for disulfide bridges with another Cys.Pro may be introduced because of its particularly planar structure,which induces β-turns in the protein's structure.

The genes encoding tankyrase derivatives and analogs of the inventioncan be produced by various methods known in the art. The manipulationswhich result in their production can occur at the gene or protein level.For example, a tankyrase gene sequence can be produced from a nativetankyrase clone by any of numerous strategies known in the art [Sambrooket al., 1989, supra]. The sequence can be cleaved at appropriate siteswith restriction endonuclease(s), followed by further enzymaticmodification if desired, isolated, and ligated in vitro. In theproduction of the gene encoding a derivative or analog of a tankyrase,care should be taken to ensure that the modified gene remains within thesame translational reading frame as the tankyrase gene, uninterrupted bytranslational stop signals, in the gene region where the desiredactivity is encoded.

Additionally, the tankyrase-encoding nucleic acid sequence can beproduced by in vitro or in vivo mutations, to create and/or destroytranslation, initiation, and/or termination sequences, or to createvariations in coding regions and/or form new restriction endonucleasesites or destroy preexisting ones, to facilitate further in vitromodification. Preferably such mutations will further enhance thespecific properties of the tankyrase gene product. Any technique formutagenesis known in the art can be used, including but not limited to,in vitro site-directed mutagenesis [Hutchinson, C., et al., J. Biol.Chem., 253:6551 (1978); Zoller and Smith, DNA, 3:479-488 (1984);Oliphant et al., Gene, 44:177 (1986); Hutchinson et al., Proc. Natl.Acad. Sci. U.S.A., 83:710 (1986)], use of TAB® linkers (Pharmacia), etc.PCR techniques are preferred for site directed mutagenesis (see Higuchi,1989, “Using PCR to Engineer DNA”, in PCR Technology: Principles andApplications for DNA Amplification, H. Erlich, ed., Stockton Press,Chapter 6, pp. 61-70). A general method for site-specific incorporationof unnatural amino acids into proteins is described in Christopher J.Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz,Science, 244:182-188 (April 1989). This method may be used to createanalogs with unnatural amino acids.

The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Examples of vectors include, but arenot limited to, E. coli, bacteriophages such as lambda derivatives, orplasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g.,pGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vectorcan, for example, be accomplished by ligating the DNA fragment into acloning vector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. Recombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation, etc., so thatmany copies of the gene sequence are generated. Preferably, the clonedgene is contained on a shuttle vector plasmid, which provides forexpansion in a cloning cell, e.g., E. coli, and facile purification forsubsequent insertion into an appropriate expression cell line, if suchis desired. For example, a shuttle vector, which is a vector that canreplicate in more than one type of organism, can be prepared forreplication in both E. coli and Saccharomyces cerevisiae by linkingsequences from an E. coli plasmid with sequences from the yeast 2 μplasmid.

In an alternative method, the desired gene may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired gene, for example, by sizefractionation, can be done before insertion into the cloning vector.

Expression of Tankyrase Polypeptides

The nucleotide sequence coding for a tankyrase, or a tankyrase relatedprotein, or a functionally equivalent derivative including a chimericprotein thereof, can be inserted into an appropriate expression vector,i.e., a vector which contains the necessary elements for thetranscription and translation of the inserted protein-coding sequence.Such elements are termed herein a “promoter.” Thus, the nucleic acidencoding a tankyrase of the invention is operationally associated with apromoter in an expression vector of the invention. Both cDNA and genomicsequences can be cloned and expressed under control of such regulatorysequences. An expression vector also preferably includes a replicationorigin.

The necessary transcriptional and translational signals can be providedon a recombinant expression vector, or they may be supplied by thenative gene encoding the corresponding tankyrase and/or its flankingregions. Any person with skill in the art of molecular biology orprotein chemistry, in view of the present disclosure, would readily knowhow to assay the protein expressed as described herein, to determinewhether such a modified protein is indeed a tankyrase. Potentialhost-vector systems include but are not limited to mammalian cellsystems infected with virus (e.g., vaccinia virus, adenovirus, etc.);insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

A recombinant tankyrase of the invention or a tankyrase-related protein,or functionally equivalent derivative, or chimeric construct may beexpressed chromosomally, after integration of the coding sequence byrecombination. In this regard, any of a number of amplification systemsmay be used to achieve high levels of stable gene expression [SeeSambrook et al., 1989, supra]. Chromosomal integration, e.g., byhomologous recombination is desirable where permanent expression isrequired, such as to immortalize an antibody-producing plasma cell. Inother embodiments, such as for in vitro propagation of cells fortransplantation, transient transfection such as with a plasmid, ispreferable. This way, the cell can be propagated indefinitely in vitro,but will terminally differentiate when reintroduced in vivo.

The cell containing the recombinant vector comprising the nucleic acidencoding a tankyrase is cultured in an appropriate cell culture mediumunder conditions that provide for expression of the tankyrase by thecell.

Any of the methods previously described for the insertion of DNAfragments into a cloning vector may be used to construct expressionvectors containing a gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombination (genetic recombination).

Expression of a tankyrase or tankyrase-related protein may be controlledby any promoter/enhancer element known in the art, but these regulatoryelements must be functional in the host selected for expression.Promoters which may be used to control tankyrase gene expressioninclude, but are not limited to, the SV40 early promoter region [Benoistand Chambon, Nature, 290:304-310 (1981)], the promoter contained in the3′ long terminal repeat of Rous sarcoma virus [Yamamoto, et al., Cell,22:787-797 (1980)], the herpes thymidine kinase promoter [Wagner et al.,Proc. Natl. Acad. Sci. U.S.A., 78:1441 -1445 (1981)], the regulatorysequences of the metallothionein gene [Brinster et al., Nature,296:39-42 (1982)]; prokaryotic expression vectors such as theβ-lactamase promoter [Villa-Kamaroff, et al., Proc. Natl. Acad. Sci.U.S.A., 75:3727-3731 (1978)], or the tac promoter [DeBoer, et al., Proc.Natl. Acad. Sci. U.S.A., 80:21-25 (1983)]; see also “Useful proteinsfrom recombinant bacteria” in Scientific American, 242:74-94 (1980);promoter elements from yeast or other fungi such as the Gal 4 promoter,the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phosphatase promoter; and the animal transcriptionalcontrol regions, which exhibit tissue specificity and have been utilizedin transgenic animals: elastase I gene control region which is active inpancreatic acinar cells [Swift et al., Cell, 38:639-646 (1984); Ornitzet al., Cold Spring Harbor Symp. Quant. Biol., 50:399-409 (1986);MacDonald, Hepatology, 7:425-515 (1987)]; insulin gene control regionwhich is active in pancreatic beta cells [Hanahan, Nature, 315:115-122(1985)], immunoglobulin gene control region which is active in lymphoidcells [Grosschedl et al., Cell, 38:647-658 (1984); Adames et al.,Nature, 318:533-538 (1985); Alexander et al., Mol. Cell. Biol.,7:1436-1444 (1987)], mouse mammary tumor virus control region which isactive in testicular, breast, lymphoid and mast cells [Leder et al.,Cell, 45:485-495 (1986)], albumin gene control region which is active inliver [Pinkert et al., Genes and Devel., 1:268-276 (1987)],alpha-fetoprotein gene control region which is active in liver [Krumlaufet al., Mol. Cell. Biol., 5:1639-1648 (1985); Hammer et al., Science,235:53-58 (1987)], alpha 1-antitrypsin gene control region which isactive in the liver [Kelsey et al., Genes and Devel., 1:161-171 (1987)],beta-globin gene control region which is active in myeloid cells [Mogramet al., Nature, 315:338-340 (1985); Kollias et al., Cell, 46:89-94(1986)], myelin basic protein gene control region which is active inoligodendrocyte cells in the brain [Readhead et al., Cell, 48:703-712(1987)], myosin light chain-2 gene control region which is active inskeletal muscle [Sani, Nature, 314:283-286 (1985)], and gonadotropicreleasing hormone gene control region which is active in thehypothalamus [Mason et al., Science, 234:1372-1378 (1986)].

Expression vectors containing a nucleic acid encoding a tankyrase of theinvention can be identified by many means including by four generalapproaches: (a) PCR amplification of the desired plasmid DNA or specificmRNA, (b) nucleic acid hybridization, (c) presence or absence ofselection marker gene functions, and (d) expression of insertedsequences. In the first approach, the nucleic acids can be amplified byPCR to provide for detection of the amplified product. In the secondapproach, the presence of a foreign gene inserted in an expressionvector can be detected by nucleic acid hybridization using probescomprising sequences that are homologous to an inserted marker gene. Inthe third approach, the recombinant vector/host system can be identifiedand selected based upon the presence or absence of certain “selectionmarker” gene functions (e.g., β-galactosidase activity, thymidine kinaseactivity, resistance to antibiotics, transformation phenotype, occlusionbody formation in baculovirus, etc.) caused by the insertion of foreigngenes in the vector. In another example, if the nucleic acid encoding atankyrase is inserted within the “selection marker” gene sequence of thevector, recombinants containing the tankyrase insert can be identifiedby the absence of the tankyrase gene function. In the fourth approach,recombinant expression vectors can be identified by assaying for theactivity, biochemical, or immunological characteristics of the geneproduct expressed by the recombinant, provided that the expressedprotein assumes a functionally active conformation, i.e., the ability oftankyrase to bind TRF1.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol El, pCR1, pBR322, pMal-C2, pET, pGEX [Smith et al., Gene, 67:31-40(1988)], pMB9 and their derivatives, plasmids such as RP4; phage DNAS,e.g., the numerous derivatives of phage λ, e.g., NM989, and other phageDNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmidssuch as the 2 μ plasmid or derivatives thereof; vectors useful ineukaryotic cells, such as vectors useful in insect or mammalian cells;vectors derived from combinations of plasmids and phage DNAs, such asplasmids that have been modified to employ phage DNA or other expressioncontrol sequences; and the like.

For example, in a baculovirus expression systems, both non-fusiontransfer vectors, such as but not limited to pVL941 (BamH1 cloning site;Summers), pVL1393 (BamH1, SmaI, XbaI, EcoR1, NotI, XmaIII, BglII, andPstI cloning site; Invitrogen), pVL1392 (BglII, PstI, NotI, XmaIII,EcoRI, XbaI, SmaI, and BamH1 cloning site; Summers and Invitrogen), andpBlueBacIII (BamH1, BglII, PstI, NcoI, and HindIII cloning site, withblue/white recombinant screening possible; Invitrogen), and fusiontransfer vectors, such as but not limited to pAc700 (BamH1 and KpnIcloning site, in which the BamH1 recognition site begins with theinitiation codon; Summers), pAc701 and pAc702 (same as pAc700, withdifferent reading frames), pAc360 (BamH1 cloning site 36 base pairsdownstream of a polyhedrin initiation codon; Invitrogen(195)), andpBlueBacHisA, B, C (three different reading frames, with BamH1, BglII,PstI, NcoI, and HindIII cloning site, an N-terminal peptide for ProBondpurification, and blue/white recombinant screening of plaques;Invitrogen (220)) can be used.

Mammalian expression vectors contemplated for use in the inventioninclude vectors with inducible promoters, such as the dihydrofolatereductase (DHFR) promoter, e.g., any expression vector with a DHFRexpression vector, or a DHFR/methotrexate co-amplification vector, suchas pED (PstI, SalI, SbaI, SmaI, and EcoRI cloning site, with the vectorexpressing both the cloned gene and DHFR; see Kaufman, Current Protocolsin Molecular Biology, 16.12 (1991). Alternatively, a glutaminesynthetase/methionine sulfoximine co-amplification vector, such as pEE14(HindIII, XbaI, SmaI, SbaI, EcoRI, and BclI cloning site, in which thevector expresses glutamine synthase and the cloned gene; Celltech). Inanother embodiment, a vector that directs episomal expression undercontrol of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamH1,SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site,constitutive RSV-LTR promoter, hygromycin selectable marker;Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII,and KpnI cloning site, constitutive hCMV immediate early gene,hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI, NheI,HindII, NotI, XhoI, SfiI, BamH1 cloning site, inducible methallothioneinIIa gene promoter, hygromycin selectable marker: Invitrogen), pREP8(BamH1, XhoI, NotI, HindIII, NheI, and KpnI cloning site, RSV-LTRpromoter, histidinol selectable marker; Invitrogen), pREP9 (KpnI, NheI,HindIII, NotI, XhoI, SfiI, and BamHI cloning site, RSV-LTR promoter,G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter,hygromycin selectable marker, N-terminal peptide purifiable via ProBondresin and cleaved by enterokinase; Invitrogen). Selectable mammalianexpression vectors for use in the invention include pRc/CMV (HindIII,BstXI, NotI, SbaI, and ApaI cloning site, G418 selection; Invitrogen),pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site, G418 selection;Invitrogen), and others. Vaccinia virus mammalian expression vectors(see, Kaufman, 1991, supra) for use according to the invention includebut are not limited to pSC11 (SmaI cloning site, TK- and β-galselection), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamHI, ApaI, NheI,SacII, KpnI, and HindIII cloning site; TK- and β-gal selection), andpTKgptF1S (EcoRI, PstI, SalI, AccI, HindII, SbaI, BamHI, and Hpa cloningsite, TK or XPRT selection).

Yeast expression systems can also be used according to the invention toexpress the tankyrase protein. For example, the non-fusion pYES2 vector(XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, Kpn1, andHindIII cloning sit; Invitrogen) or the fusion pYESHisA, B, C (XbaI,SphI, ShoI, NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloningsite, N-terminal peptide purified with ProBond resin and cleaved withenterokinase; Invitrogen), to mention just two, can be employedaccording to the invention.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification (e.g., glycosylation,cleavage [e.g., of signal sequence]) of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce an non-glycosylated coreprotein product. Expression in yeast can produce a glycosylated product.Expression in eukaryotic cells can increase the likelihood of “native”glycosylation and folding of a heterologous protein. Moreover,expression in mammalian cells can provide a tool for reconstituting, orconstituting, the tankyrase activity. Furthermore, different vector/hostexpression systems may affect processing reactions, such as proteolyticcleavages, to a different extent.

Vectors are introduced into the desired host cells by methods known inthe art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter [see, e.g., Wu et al., J. Biol. Chem. 267:963-967(1992); Wu and Wu, J. Biol. Chem., 263:14621-14624 (1988); Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

Gene Therapy and Transgenic Vectors

A gene encoding a tankyrase or derivative thereof, including an inactivederivative can be introduced either in vivo, ex vivo, or in vitro in aviral vector. Such vectors include an attenuated or defective DNA virus,such as but not limited to herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. Defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. For example, in the treatment ofataxia telangiectasia, T lymphocytes can be specifically targeted.Examples of particular vectors include, but are not limited to, adefective herpes virus 1 (HSV1) vector [Kaplitt et al., Molec. Cell.Neurosci., 2:320-330 (1991)], an attenuated adenovirus vector, such asthe vector described by Stratford-Perricaudet et al. [J. Clin. Invest.,90:626-630 (1992)], and a defective adeno-associated virus vector[Samulski et al., J. Virol., 61:3096-3101 (1987); Samulski et al., J.Virol., 63:3822-3828 (1989)].

Preferably, for in vitro administration, an appropriateimmunosuppressive treatment is employed in conjunction with the viralvector, e.g., adenovirus vector, to avoid immuno-deactivation of theviral vector and transfected cells. For example, immunosuppressivecytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), oranti-CD4 antibody, can be administered to block humoral or cellularimmune responses to the viral vectors [see, e.g., Wilson, NatureMedicine, (1995)]. In addition, it is advantageous to employ a viralvector that is engineered to express a minimal number of antigens.

In another embodiment the gene can be introduced in a retroviral vector,e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346; Mann etal., Cell, 33:153 (1983); Temin et al., U.S. Pat. No. 4,650,764; Teminet al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol., 62:1120(1988); Temin et al., U.S. Pat. No. 5,124,263; International PatentPublication No. WO 95/07358, published Mar. 16, 1995, by Dougherty etal.; and Kuo et al., Blood, 82:845 (1993).

Targeted gene delivery is described in International Patent PublicationWO 95/28494, published October 1995.

Alternatively, the vector can be introduced by lipofection. For the pastdecade, there has been increasing use of liposomes for encapsulation andtransfection of nucleic acids in vitro. Synthetic cationic lipidsdesigned to limit the difficulties and dangers encountered with liposomemediated transfection can be used to prepare liposomes for in vivotransfection of a gene encoding a marker [Felgner, et. al., Proc. Natl.Acad. Sci. U.S.A., 84:7413-7417 (1987); see Mackey, et al., Proc. Natl.Acad. Sci. U.S.A., 85:8027-8031 (1988)]. The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes [Felgner andRingold, Science, 337:387-388 (1989)]. The use of lipofection tointroduce exogenous genes into the specific organs in vivo has certainpractical advantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly advantageous in a tissuewith cellular heterogeneity, such as pancreas, liver, kidney, and thebrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting [see Mackey, et. al., 1988, supra]. Targetedpeptides, e.g., hormones or neurotransmitters, and proteins such asantibodies, or non-peptide molecules could be coupled to liposomeschemically.

It is also possible to introduce the vector as a naked DNA plasmid.Naked DNA vectors for gene therapy can be introduced into the desiredhost cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter [see, e.g., Wu et al., J. Biol. Chem.,267:963-967 (1992); Wu and Wu, J. Biol. Chem., 263:14621-14624 (1988);Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar.15, 1990].

In a further embodiment, the present invention provides forco-expression of tankyrase and a TRF1 and/or a TRF1 enhancing gene undercontrol of a specific DNA recognition sequence by providing a genetherapy expression vector comprising a tankyrase coding gene, and a TRF1coding gene and/or a TRF1 enhancing gene under control of, inter alia, aTRF1 regulatory sequence. In one embodiment, these elements are providedon separate vectors.

General Protein Purification Procedures

Initial steps for purifying the tankyrase of the present invention caninclude salting in or salting out, such as in ammonium sulfatefractionations; solvent exclusion fractionations, e.g., an ethanolprecipitation; detergent extractions to free membrane bound proteinsusing suchdetergents as TRITON X-100, TWEEN-20 etc.; or high saltextractions. Solubilization of proteins may also be achieved usingaprotic solvents such as dimethyl sulfoxide and hexamethylphosphoramide.In addition, high speed ultracentrifugation may be used either alone orin conjunction with other extraction techniques.

Generally good secondary isolation or purification steps include solidphase absorption using calcium phosphate gel or hydroxyapatite; or solidphase binding. Solid phase binding may be performed through ionicbonding, with either an anion exchanger, such as diethylaminoethyl(DEAE), or diethyl [2-hydroxypropyl] aminoethyl (QAE) SEPHADEX orcellulose; or with a cation exchanger such as carboxymethyl (CM) orsulfopropyl (SP) SEPHADEX or cellulose. Alternative means of solid phasebinding includes the exploitation of hydrophobic interactions e.g., theusing of a solid support such as phenylSepharose and a high salt buffer;affinity-binding, using, e.g., placing the N-terminal acidic domain ofTRF1 on an activated support; immuno-binding, using e.g., an antibody toa tankyrase bound to an activated support; as well as other solid phasesupports including those that contain specific dyes or lectins etc. Afurther solid phase support technique that is often used at the end ofthe purification procedure relies on size exclusion, such as SEPHADEXand SEPHAROSE gels, or pressurized or centrifugal membrane techniques,using size exclusion membrane filters.

Solid phase support separations are generally performed batch-wise withlow-speed centrifugations or by column chromatography. High performanceliquid chromatography (HPLC), including such related techniques as FPLC,is presently the most common means of performing liquid chromatography.Size exclusion techniques may also be accomplished with the aid of lowspeed centrifugation.

In addition size permeation techniques such as gel electrophoretictechniques may be employed. These techniques are generally performed intubes, slabs or by capillary electrophoresis.

Almost all steps involving protein purification employ a bufferedsolution. Unless otherwise specified, generally 25-100 mM concentrationsof buffer salts are used. Low concentration buffers generally imply 5-25mM concentrations. High concentration buffers generally implyconcentrations of the buffering agent of between 0.1-2 M concentrations.Typical buffers can be purchased from most biochemical catalogues andinclude the classical buffers such as Tris, pyrophosphate, monophosphateand diphosphate and the Good buffers [Good, N. E., et al., Biochemistry,5:467 (1966); Good, N. E. and Izawa, S., Meth. Enzymol., 24B:53 (1972);and Fergunson, W. J. and Good, N. E., Anal. Biochem., 104:300 (1980]such as Mes, Hepes, Mops, tricine and Ches.

Materials to perform all of these techniques are available from avariety of sources such as Sigma Chemical Company in St. Louis, Mo.

Antibodies to the Tankyrases

According to the present invention, the tankyrase or tankyrase-relatedproteins as produced by a recombinant source, or through chemicalsynthesis, or a tankyrase or tankyrase-related protein isolated fromnatural sources; and derivatives or analogs thereof, including fusionproteins, may be used as an immunogen to generate antibodies thatrecognize the tankyrase or tankyrase-related protein, as exemplifiedbelow. Such antibodies include but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments, and a Fab expressionlibrary. The anti-tankyrase antibodies of the invention may be crossreactive, that is, they may recognize a tankyrase derived from adifferent source. Polyclonal antibodies have greater likelihood of crossreactivity. Alternatively, an antibody of the invention may be specificfor a single form of a tankyrase, such as the tankyrase having an aminoacid sequence of SEQ ID NO:2.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to tankyrase or derivative or analog thereof. Forthe production of antibody, various host animals can be immunized byinjection with the tankyrase, or a derivative (e.g., or fusion protein)thereof, including but not limited to rabbits, mice, rats, sheep, goats,etc. In one embodiment, the tankyrase can be conjugated to animmunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpethemocyanin (KLH). Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the tankyrase,or analog, or derivative thereof, any technique that provides for theproduction of antibody molecules by continuous cell lines in culture maybe used. These include but are not limited to the hybridoma techniqueoriginally developed by Kohler and Milstein [Nature, 256:495-497(1975)], as well as the trioma technique, the human B-cell hybridomatechnique [Kozbor et al., Immunology Today, 4:72 (1983); Cote et al.,Proc. Natl. Acad. Sci. U.S.A., 80:2026 -2030 (1983)], and theEBV-hybridoma technique to produce human monoclonal antibodies [Cole etal., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96 (1985)]. In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals utilizingrecent technology [PCT/US90/02545]. In fact, according to the invention,techniques developed for the production of “chimeric antibodies”[Morrison et al., J. Bacteriol., 159:870 (1984); Neuberger et al.,Nature, 312:604-608 (1984); Takeda et al., Nature, 314:452-454 (1985)]by splicing the genes from a mouse antibody molecule specific for atankyrase together with genes from a human antibody molecule ofappropriate biological activity can be used; such antibodies are withinthe scope of this invention. Such human or humanized chimeric antibodiesare preferred for use in therapy of human diseases or disorders(described infra), since the human or humanized antibodies are much lesslikely than xenogenic antibodies to induce an immune response, inparticular an allergic response, themselves.

According to the invention, techniques described for the production ofsingle chain antibodies [U.S. Pat. Nos. 5,476,786 and 5,132,405 toHuston; U.S. Pat. No. 4,946,778] can be adapted to produce e.g.,tankyrase-specific single chain antibodies. An additional embodiment ofthe invention utilizes the techniques described for the construction ofFab expression libraries [Huse et al., Science, 246:1275-1281 (1989)] toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity for an A-tankyrase, or its derivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), Western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of tankyrase, one may assay generated hybridomas for aproduct which binds to the tankyrase fragment containing such epitopeand choose those which do not cross-react with tankyrase. For selectionof an antibody specific to a tankyrase from a particular source, one canselect on the basis of positive binding with tankyrase expressed by orisolated from that specific source.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the tankyrase, e.g., forWestern blotting, imaging tankyrase in situ, measuring levels thereof inappropriate physiological samples, etc. using any of the detectiontechniques mentioned herein or known in the art.

In a specific embodiment, antibodies that agonize or antagonize theactivity of tankyrase can be generated. Such antibodies can be testedusing the assays described infra for identifying ligands.

Labels

The tankyrases of the present invention, antibodies to tankyrases,nucleic acids that hybridize to SEQ ID NO:1 (e.g. probes), as well asnucleic acids that comprise the specific nucleotide sequences thattankyrases bind, can all be labeled. Suitable labels include enzymes,fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE), Texas red (TR), rhodamine, free or chelated lanthanide seriessalts, especially Eu³⁺, to name a few fluorophores), chromophores,radioisotopes, chelating agents, dyes, colloidal gold, latex particles,ligands (e.g., biotin), and chemiluminescent agents. When a controlmarker is employed, the same or different labels may be used for thereceptor and control marker.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, 125I, ¹³¹I, and ¹⁸⁶Re areused, known currently available counting procedures may be utilized.Such labels may also be appropriate for the nucleic acid probes used inbinding studies with tankyrase. In the instance where the label is anenzyme, detection may be accomplished by any of the presently utilizedcolorimetric, spectrophotometric, fluorospectrophotometric, amperometricor gasometric techniques known in the art.

Direct labels are one example of labels which can be used according tothe present invention. A direct label has been defined as an entity,which in its natural state, is readily visible, either to the naked eye,or with the aid of an optical filter and/or applied stimulation, e.g.U.V. light to promote fluorescence. Among examples of colored labels,which can be used according to the present invention, include metallicsol particles, for example, gold sol particles such as those describedby Leuvering (U.S. Pat. No. 4,313,734); dye sole particles such asdescribed by Gribnau et al. (U.S. Pat. No. 4,373,932) and May et al. (WO88/08534); dyed latex such as described by May, supra, Snyder (EP-A 0280 559 and 0 281 327); or dyes encapsulated in liposomes as describedby Campbell et al. (U.S. Pat. No. 4,703,017). Other direct labelsinclude a radionucleotide, a fluorescent moiety or a luminescent moiety.In addition to these direct labeling devices, indirect labels comprisingenzymes can also be used according to the present invention. Varioustypes of enzyme linked immunoassays are well known in the art, forexample, alkaline phosphatase and horseradish peroxidase, lysozyme,glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, theseand others have been discussed in detail by Eva Engvall in EnzymeImmunoassay ELISA and EMIT in Methods in Enzymology, 70:419-439 (1980)and in U.S. Pat. No. 4,857,453.

Suitable enzymes include, but are not limited to, alkaline phosphataseand horseradish peroxidase.

In addition, a tankyrase or fragment thereof can be modified to containa marker protein such as green fluorescent protein as described in U.S.Pat. No. 5,625,048 filed Apr. 29, 1997 and WO 97/26333, published Jul.24, 1997 each of which are hereby incorporated by reference herein intheir entireties.

Other labels for use in the invention include magnetic beads or magneticresonance imaging labels.

In another embodiment, a phosphorylation site can be created on anantibody of the invention for labeling with ³²P, e.g., as described inEuropean Patent No. 0372707 (application No. 89311108.8) by SidneyPestka, or U.S. Pat. No. 5,459,240, issued Oct. 17, 1995 to Foxwell etal.

As exemplified herein, proteins, including antibodies, can be labeled bymetabolic labeling. Metabolic labeling occurs during in vitro incubationof the cells that express the protein in the presence of culture mediumsupplemented with a metabolic label, such as [³⁵S]-methionine or[³²P]-orthophosphate. In addition to metabolic (or biosynthetic)labeling with [³⁵S]-methionine, the invention further contemplateslabeling with [¹⁴C]-amino acids and [³H]-amino acids (with the tritiumsubstituted at non-labile positions).

Gene Therapy and Transgenic Vectors

In one embodiment, a gene encoding a tankyrase or structural/functionaldomain thereof is introduced in vivo in a viral vector. Such vectorsinclude an attenuated or defective DNA virus, such as but not limited toherpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV),adenovirus, adeno-associated virus (AAV), and the like. Defectiveviruses, which entirely or almost entirely lack viral genes, arepreferred. Defective virus is not infective after introduction into acell. Use of defective viral vectors allows for administration to cellsin a specific, localized area, without concern that the vector caninfect other cells. Thus, any tissue can be specifically targeted.Examples of particular vectors include, but are not limited to, adefective herpes virus 1 (HSV1) vector [Kaplitt et al., Molec. Cell.Neurosci. 2:320-330 (1991)], an attenuated adenovirus vector, such asthe vector described by Stratford-Perricaudet et al. [J. Clin. Invest.90:626-630 (1992)], and a defective adeno-associated virus vector[Samulski et al., J. Virol. 61:3096-3101 (1987); Samulski et al., J.Virol. 63:3822-3828 (1989)].

Preferably, for in vitro administration, an appropriateimmunosuppressive treatment is employed in conjunction with the viralvector, e.g., adenovirus vector, to avoid immuno-deactivation of theviral vector and transfected cells. For example, immunosuppressivecytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), oranti-CD4 antibody, can be administered to block humoral or cellularimmune responses to the viral vectors [see, e.g., Wilson, NatureMedicine (1995)]. In addition, it is advantageous to employ a viralvector that is engineered to express a minimal number of antigens.

In another embodiment the gene can be introduced in a retroviral vector,e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346; Mann etal., 1983, Cell 33:153; Temin et al., U.S. Pat. No. 4,650,764; Temin etal., U.S. Pat. No. 4,980,289; Markowitz et al., 1988, J. Virol. 62:1120;Temin et al., U.S. Pat. No. 5,124,263; International Patent PublicationNo. WO 95/07358, published Mar. 16, 1995, by Dougherty et al.; and Kuoet al., 1993, Blood 82:845.

Targeted gene delivery is described in International Patent PublicationWO 95/28494, published October 1995.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker [Felgner,et. al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417 (1987); see Mackey,et al., Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031 (1988)]. The use ofcationic lipids may promote encapsulation of negatively charged nucleicacids, and also promote fusion with negatively charged cell membranes[Felgner and Ringold, Science 337:387-388 (1989)]. The use oflipofection to introduce exogenous genes into the specific organs invivo has certain practical advantages. Molecular targeting of liposomesto specific cells represents one area of benefit. It is clear thatdirecting transfection to particular cell types would be particularlyadvantageous in a tissue with cellular heterogeneity, such as pancreas,liver, kidney, and the brain. Lipids may be chemically coupled to othermolecules for the purpose of targeting [see Mackey, et. al., supra].Targeted peptides, e.g., hormones or neurotransmitters, and proteinssuch as antibodies, or non-peptide molecules could be coupled toliposomes chemically.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter [see, e.g., Wu et al., J. Biol. Chem. 267:963-967(1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990].

In a preferred embodiment of the present invention, a gene therapyvector as described above employs a transcription control sequenceoperably associated with the sequence for the tankyrase inserted in thevector. That is, a specific expression vector of the present inventioncan be used in gene therapy.

Such an expression vector is particularly useful to regulate expressionof a therapeutic tankyrase gene. In one embodiment, the presentinvention contemplates constitutive expression of the tankyrase gene,even if at low levels. In general, see U.S. Pat. No. 5,399,346 toAnderson et al.

Antisense, Gene Targeting and Ribozymes

The functional activity of tankyrase can be evaluated transgenically. Inthis respect, a transgenic mouse model can be used. The tankyrase genecan be used in complementation studies employing transgenic mice.Transgenic vectors, including viral vectors, or cosmid clones (or phageclones) corresponding to the wild type locus of candidate gene, can beconstructed using the isolated tankyrase gene. Cosmids may be introducedinto transgenic mice using published procedures [Jaenisch, Science,240:1468-1474 (1988)]. In a genetic sense, the transgene acts as asuppressor mutation.

Alternatively, a transgenic animal model can be prepared in whichexpression of the tankyrase gene is disrupted. Gene expression isdisrupted, according to the invention, when no functional protein isexpressed. One standard method to evaluate the phenotypic effect of agene product is to employ knock-out technology to delete a gene asdescribed in U.S. Pat. 5,464,764, Issued Nov. 7, 1995; and U.S. Pat. No.5,777,195, Issued Jul. 7, 1998 (both of which are hereby incorporated byreference herein in their entireties.)

The present invention also extends to the preparation of antisensenucleotides and ribozymes that may be used to interfere with theexpression of tankyrase at the translational level. This approachutilizes antisense nucleic acid and ribozymes to block translation of aspecific mRNA, either by masking that mRNA with an antisense nucleicacid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule [See Weintraub, Sci.Amer. 262:40-46 (1990); Marcus-Sekura, Nucl. Acid Res, 15: 5749-5763(1987); Marcus-Sekura Anal.Biochem., 172:289-295 (1988); Brysch et al.,Cell Mol. Neurobiol., 14:557-568 (1994)]. Preferably, the antisensemolecule employed is complementary to a substantial portion of the mRNA.In the cell, the antisense molecule hybridizes to that mRNA, forming adouble stranded molecule. The cell does not translate an mRNA in thisdouble-stranded form. Therefore, antisense nucleic acids interfere withthe expression of mRNA into protein. Preferably a DNA antisense nucleicacid is employed since such an RNA/DNA duplex is a preferred substratefor RNase H. Oligomers of greater than about fifteen nucleotides andmolecules that hybridize to the AUG initiation codon will beparticularly efficient, though larger molecules that are essentiallycomplementary to the entire mRNA are more likely to be effective.Antisense methods have been used to inhibit the expression of many genesin vitro [Marcus-Sekura, Anal.Biochem., 172:289-295 (1988); Hambor etal., Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014 (1988)] and in situ[Arima et al., Antisense Nucl. Acid Drug Dev. 8:319-327 (1998); Hou etal., Antisense Nucl. Acid Drug Dev. 8:295-308 (1998); U.S. Pat. No.5,726,020, Issued Mar. 10, 1998; and U.S. Pat. No. 5,731,294, IssuedMar. 24, 1998, all of which are incorporated by reference in theirentireties].

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these ribozymes,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it [Cech, JAMA,260:3030-3034 (1988); Cech, Biochem. Intl, 18:7-14 (1989)]. Because theyare sequence-specific, only mRNAs with particular sequences areinactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand “hammerhead”-type [Haselhoff and Gerlach, Nature 334:585-591(1988)]. Tetrahymena-type ribozymes recognize four-base sequences, while“hammerhead”-type recognize eleven- to eighteen-base sequences. Thelonger the recognition sequence, the more likely it is to occurexclusively in the target mRNA species. Therefore, hammerhead-typeribozymes are preferable to Tetrahymena-type ribozymes for inactivatinga specific mRNA species, and eighteen base recognition sequences arepreferable to shorter recognition sequences.

The DNA sequences described herein may thus be used to prepare antisensemolecules against, and ribozymes that cleave mRNAs for tankyrase andtheir ligands.

Kits

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of predetermined telomere-binding activity orpredetermined telomere lengthening activity capability in suspectedtarget cells. In accordance with the testing techniques discussed above,one class of such kits will contain at least the labeled tankyrase orits binding partner, for instance an antibody specific thereto, anddirections, of course, depending upon the method selected, e.g.,“competitive”, “sandwich”, “DASP”, and the like. The kits may alsocontain peripheral reagents such as buffers, stabilizers, etc. and/ordirections.

Drug Screens

In addition to rational design of agonists and antagonists based on thestructure of tankyrase the present invention further contemplates analternative method for identifying specific antagonists or agonistsusing various screening assays known in the art.

Accordingly any screening technique known in the art can be used toscreen for agonists or antagonists to tankyrase. The present inventioncontemplates screens for small molecule ligands or ligand analogs andmimics, as well as screens for natural ligands that bind to and agonizeor antagonize tankyrase in vivo. For example, natural products librariescan be screened using assays of the invention for molecules that agonizeor antagonize tankyrase activity.

For example, the present invention provides methods of identifyingagents that modulate the poly (ADP-ribose) polymerase activity of thetankyrases of the present invention. In a particular embodiment, thepoly (ADP-ribose) polymerase activity is determined using α-³²PNAD⁺, aprotein substrate for the tankyrase (such as a histone, or TRF1, orfragment thereof), a tankyrase (or a fragment thereof containing anactive PARP domain) in the presence and absence of potential agonistsand/or antagonists. The PARP activity can be determined as a function ofthe amount of ³²P labeled protein substrate generated. Alternatively,cold NAD⁺ can be used and the labeled protein substrate can bedetermined using an antibody that is specific for PARP labeled proteins.In one embodiment, the protein substrate is placed on a nitrocellularfilter and the assay is an activity blot [Simonin et al., J. Biol.Chem., 265:19249-19256 (1990)]. In another embodiment the labeledprotein substrate is precipitated (e.g. by trichloroacetic) and/orplaced on an SDS gel following a solution assay [Simonin et al., J.Biol. Chem., 268:13454-13461 (1993)].

Knowledge of the primary sequence of tankyrase and the similarity ofseveral domains with those contained in other proteins, can also provideclue as the inhibitors or antagonists of the protein. Identification andscreening of antagonists is further facilitated by determiningstructural features of the protein, e.g., using X-ray crystallography,neutron diffraction, nuclear magnetic resonance spectrometry, and othertechniques for structure determination. These techniques provide for therational design or identification of agonists and antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” [Scott and Smith, 1990, Science249:386-390 (1990); Cwirla, et al., Proc. Natl. Acad. Sci., 87:6378-6382(1990); Devlin et al., Science, 249:404-406 (1990)], very largelibraries can be constructed (10⁶-10⁸ chemical entities). A secondapproach uses primarily chemical methods, of which the Geysen method[Geysen et al., Molecular Immunology 23:709-715 (1986); Geysen et al. J.Immunologic Method 102:259-274 (1987)] and the method of Fodor et al.[Science 251:767-773 (1991)] are examples. Furka et al. [14thInternational Congress of Biochemistry, Volume 5, Abstract FR:013(1988); Furka, Int. J. Peptide Protein Res. 37:487-493 (1991)], Houghton[U.S. Pat. No. 4,631,211, issued December 1986] and Rutter et al. [U.S.Pat. No. 5,010,175, issued Apr. 23, 1991] describe methods to produce amixture of peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries [Needels et al., Proc. Natl.Acad. Sci. USA 90:10700-4 (1993); Ohlmeyer et al., Proc. Natl. Acad.Sci. USA 90:10922-10926 (1993); Lam et al., International PatentPublication No. WO 92/00252; Kocis et al., International PatentPublication No. WO 9428028, each of which is incorporated herein byreference in its entirety], and the like can be used to screen forligands to the tankyrase according to the present invention.

Alternatively, assays for binding of soluble ligand to cells thatexpress recombinant forms of the tankyrase can be performed. The solubleligands can be provided readily as recombinant or syntheticpolypeptides.

The screening can be performed with recombinant cells that express atankyrase, or fragment thereof, e.g. the portion of tankyrase requiredfor binding TRF1 or alternatively, using purified protein, e.g.,produced recombinantly, as described above. For example, the ability oflabeled, soluble or solubilized tankyrase to bind TRF1 can be used toscreen libraries, as described in the foregoing references.

In one such example, a phage library can be employed. Phage librarieshave been constructed which when infected into host E. coli producerandom peptide sequences of approximately 10 to 15 amino acids [Parmleyand Smith, Gene, 73:305-318 (1988), Scott and Smith, Science,249:386-249 (1990)]. Specifically, the phage library can be mixed in lowdilutions with permissive E. coli in low melting point LB agar which isthen poured on top of LB agar plates. After incubating the plates at 37°C. for a period of time, small clear plaques in a lawn of E. coli willform which represents active phage growth and lysis of the E. coli. Arepresentative of these phages can be absorbed to nylon filters byplacing dry filters onto the agar plates. The filters can be marked fororientation, removed, and placed in washing solutions to block anyremaining absorbent sites. The filters can then be placed in a solutioncontaining, for example, a radioactive fragment of tankyrase containingthe TRF1 binding domain. After a specified incubation period, thefilters can be thoroughly washed and developed for autoradiography.Plaques containing the phage that bind to the radioactive TRF1 bindingdomain of tankyrase can then be identified. These phages can be furthercloned and then retested for their ability to hinder the binding oftankyrase to TRF1, for example. Once the phages have been purified, thebinding sequence contained within the phage can be determined bystandard DNA sequencing techniques. Once the DNA sequence is known,synthetic peptides can be generated which represents these sequences.

It an alternative embodiment, the radioactive tankyrase fragment cancontain the PARP-related domain. Plaques containing the phage that bindto the radioactive PARP-related domain can be identified, further clonedand retested for their ability to hinder the PARP activity of tankyrase.Again, once the phages have been purified, the binding sequencecontained within the phage can be determined by standard DNA sequencingtechniques. Once the DNA sequence is known, synthetic peptides can begenerated which represents these sequences.

These peptides can be tested, for example, for their ability tointerfere with tankyrase binding to TRF1, for example.

The effective peptide(s) can be synthesized in large quantities for usein in vivo models and eventually in humans to stimulate telomereelongation. It should be emphasized that synthetic peptide production isrelatively non-labor intensive, easily manufactured, quality controlledand thus, large quantities of the desired product can be produced quitecheaply. Similar combinations of mass produced synthetic peptides haverecently been used with great success [Patarroyo, Vaccine, 10:175-178(1990)].

Alternatively, known inhibitors of PARP activity can be used to inhibittankyrase activity, in situ and/or in vivo, thereby aiding in themodulation of telomere length. Telomere lengthening could be beneficialboth in the extension of the life-span of non-tumor cells, as well as inthe inhibition of tumor cell growth. Inhibitors of PARP activity areknown in the art and include 3-aminobenzamide (3ab) and relatedinhibitors [Durkaczm et al., Nature, 283:593-596 (1980); Oikawa et al.,Biochem. Biophys. Res. Commun., 97:1311-1316 (1980)].

Administration

According to the invention, the component or components of a therapeuticcomposition, e.g., a tankyrase or a tankyrase inhibitor such as3-aminobenzamide and a pharmaceutically acceptable carrier, of theinvention may be introduced parenterally, transmucosally, e.g., orally,nasally, or rectally, or transdermally. Preferably, administration isparenteral, e.g., via intravenous injection, and also including, but isnot limited to, intra-arteriole, intramuscular, intradermal,subcutaneous, intraperitoneal, intraventricular, and intracranialadministration.

In a preferred aspect, a tankyrase of the present invention can crosscellular or nuclear membranes, which would allow for intravenous or oraladministration. Strategies are available for such crossing, includingbut not limited to, increasing the hydrophobic nature of a molecule;introducing the molecule as a conjugate to a carrier, such as a ligandto a specific receptor, targeted to a receptor; and the like.

The present invention also provides for conjugating targeting moleculesto a tankyrase. “Targeting molecule” as used herein shall mean amolecule which, when administered in vivo, localizes to desiredlocation(s). In various embodiments, the targeting molecule can be apeptide or protein, antibody, lectin, carbohydrate, or steroid. In oneembodiment, the targeting molecule is a peptide ligand of a receptor onthe target cell. In a specific embodiment, the targeting molecule is anantibody. Preferably, the targeting molecule is a monoclonal antibody.In one embodiment, to facilitate crosslinking the antibody can bereduced to two heavy and light chain heterodimers, or the F(ab′)₂fragment can be reduced, and crosslinked to the tankyrase via thereduced sulfhydryl.

Antibodies for use as targeting molecule are specific for cell surfaceantigen. In one embodiment, the antigen is a receptor. For example, anantibody specific for a receptor on T lymphocyte receptor, can be usedin the treatment of ataxia telangiectasia. This invention furtherprovides for the use of other targeting molecules, such as lectins,carbohydrates, proteins and steroids.

In another embodiment, the therapeutic compound can be delivered in avesicle, in particular a liposome [see Langer, Science, 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.]. To reduce its systemic side effects, this may be a preferredmethod for introducing a tankyrase.

In yet another embodiment, the therapeutic compound can be delivered ina controlled release system. For example, the polypeptide may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used [see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng., 14:201 (1987); Buchwald et al., Surgery, 88:507(1980); Saudek et al., N. Engl. J. Med., 321:574 (1989)]. In anotherembodiment, polymeric materials can be used [see Medical Applications ofControlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Fla.(1974); Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger andPeppas, J. Macromol. Sci. Rev. Macromol. Chem., 23:61 (1983); see alsoLevy et al., Science, 228:190 (1985); During et al., Ann. Neurol.,25:351 (1989); Howard et al., J. Neurosurg., 71:105 (1989)]. In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose [see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)].Preferably, a controlled release device is introduced into a subject inproximity of the site of inappropriate immune activation or a tumor.Other controlled release systems are discussed in the review by Langer[Science, 249:1527-1533 (1990)].

Pharmaceutical Compositions.

In yet another aspect of the present invention, provided arepharmaceutical compositions of the above. Such pharmaceuticalcompositions may be for administration for injection, or for oral,pulmonary, nasal or other forms of administration. In general,comprehended by the invention are pharmaceutical compositions comprisingeffective amounts of a low molecular weight component or components, orderivative products, of the invention together with pharmaceuticallyacceptable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers. Such compositions include diluents of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength;additives such as detergents and solubilizing agents (e.g., Tween 80,Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol); incorporation of thematerial into particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronicacid may also be used. Such compositions may influence the physicalstate, stability, rate of in vivo release, and rate of in vivo clearanceof the present proteins and derivatives. See, e.g., Remington'sPharmaceutical Sciences, 18th Ed. [1990, Mack Publishing Co., Easton,Pa. 18042] pages 1435-1712 which are herein incorporated by reference.The compositions may be prepared in liquid form, or may be in driedpowder, such as lyophilized form.

Oral Delivery.

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited byG. S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated byreference. In general, the formulation will include a tankyrase (orchemically modified forms thereof) and inert ingredients which allow forprotection against the stomach environment, and release of thebiologically active material in the intestine.

Also specifically contemplated are oral dosage forms of the abovederivatized component or components. The component or components may bechemically modified so that oral delivery of the derivative isefficacious. Generally, the chemical modification contemplated is theattachment of at least one moiety to the component molecule itself,where said moiety permits (a) inhibition of proteolysis; and (b) uptakeinto the blood stream from the stomach or intestine. Also desired is theincrease in overall stability of the component or components andincrease in circulation time in the body. An example of such a moiety ispolyethylene glycol.

For the component (or derivative) the location of release may be thestomach, the small intestine (the duodenum, the jejunum, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the protein (or derivative) or byrelease of the biologically active material beyond the stomachenvironment, such as in the intestine.

The therapeutic can be included in the formulation as finemulti-particulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Binders also may be used to hold the therapeutic agenttogether to form a hard tablet and include materials from naturalproducts such as acacia, tragacanth, starch and gelatin.

An anti-frictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall. Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression also might beadded. The glidants may include starch, talc, pyrogenic silica andhydrated silicoaluminate.

In addition, to aid dissolution of the therapeutic into the aqueousenvironment a surfactant might be added as a wetting agent. Additiveswhich potentially enhance uptake of the protein (or derivative) are forinstance the fatty acids oleic acid, linoleic acid and linolenic acid.

Nasal Delivery.

Nasal delivery of a tankyrase or derivative thereof is alsocontemplated. Nasal delivery allows the passage of the protein to theblood stream directly after administering the therapeutic product to thenose, without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran.

For nasal administration, a useful device is a small, hard bottle towhich a metered dose sprayer is attached. In one embodiment, the metereddose is delivered by drawing the pharmaceutical composition of thepresent invention solution into a chamber of defined volume, whichchamber has an aperture dimensioned to aerosolize and aerosolformulation by forming a spray when a liquid in the chamber iscompressed. The chamber is compressed to administer the pharmaceuticalcomposition of the present invention. In a specific embodiment, thechamber is a piston arrangement. Such devices are commerciallyavailable.

Alternatively, a plastic squeeze bottle with an aperture or openingdimensioned to aerosolize an aerosol formulation by forming a spray whensqueezed. The opening is usually found in the top of the bottle, and thetop is generally tapered to partially fit in the nasal passages forefficient administration of the aerosol formulation. Preferably, thenasal inhaler will provide a metered amount of the aerosol formulation,for administration of a measured dose of the drug.

Transdermal Administration.

Various and numerous methods are known in the art for transdermaladministration of a drug, e.g., via a transdermal patch. Transdermalpatches are described in for example, U.S. Pat. No. 5,407,713, issuedApr. 18, 1995 to Rolando et al.; U.S. Pat. No. 5,352,456, issued Oct. 4,1004 to Fallon et al.; U.S. Pat. No. 5,332,213 issued Aug. 9, 1994 toD'Angelo et al.; U.S. Pat. No. 5,336,168, issued Aug. 9, 1994 toSibalis; U.S. Pat. No. 5,290,561, issued Mar. 1, 1994 to Farhadieh etal.; U.S. Pat. No. 5,254,346, issued Oct. 19, 1993 to Tucker et al.;U.S. Pat. No. 5,164,189, issued Nov. 17, 1992 to Berger et al.; U.S.Pat. No. 5,163,899, issued Nov. 17, 1992 to Sibalis; U.S. Pat. Nos.5,088,977 and 5,087,240, both issued Feb. 18, 1992 to Sibalis; U.S. Pat.No. 5,008,110, issued Apr. 16, 1991 to Benecke et al.; and U.S. Pat. No.4,921,475, issued May 1, 1990 to Sibalis, the disclosure of each ofwhich is incorporated herein by reference in its entirety.

It can be readily appreciated that a transdermal route of administrationmay be enhanced by use of a dermal penetration enhancer, e.g., such asenhancers described in U.S. Pat. No. 5,164,189 (supra), U.S. Pat. No.5,008,110 (supra), and U.S. Pat. No. 4,879,119, issued Nov. 7, 1989 toAruga et al., the disclosure of each of which is incorporated herein byreference in its entirety.

Pulmonary Delivery.

Also contemplated herein is pulmonary delivery of the pharmaceuticalcompositions of the present invention. A pharmaceutical composition ofthe present invention is delivered to the lungs of a mammal whileinhaling and traverses across the lung epithelial lining to the bloodstream. Other reports of this include Adjei et al. [PharmaceuticalResearch, 7:565-569 (1990); Adjei et al., International Journal ofPharmaceutics, 63:135-144 (1990) (leuprolide acetate); Braquet et al.,Journal of Cardiovascular Pharmacology, 13(suppl. 5):143-146 (1989)(endothelin-1); Hubbard et al., Annals of Internal Medicine, Vol. III,pp. 206-212 (1989) (α1-antitrypsin); Smith et al., J. Clin. Invest.,84:1145-1146 (1989) (α-1-proteinase); Oswein et al., “Aerosolization ofProteins”, Proceedings of Symposium on Respiratory Drug Delivery II,Keystone, Co., March, (1990) (recombinant human growth hormone); Debs etal., J. Immunol., 140:3482-3488 (1988) (interferon-γ and tumor necrosisfactor alpha); Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colonystimulating factor)]. A method and composition for pulmonary delivery ofdrugs for systemic effect is described in U.S. Pat. No. 5,451,569,issued Sep. 19, 1995 to Wong et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. With regard to construction of the delivery device,any form of aerosolization known in the art, including but not limitedto spray bottles, nebulization, atomization or pump aerosolization of aliquid formulation, and aerosolization of a dry powder formulation, canbe used in the practice of the invention.

All such devices require the use of formulations suitable for thedispensing of pharmaceutical composition of the present invention (orderivative). Typically, each formulation is specific to the type ofdevice employed and may involve the use of an appropriate propellantmaterial, in addition to the usual diluents, adjuvants and/or carriersuseful in therapy. Also, the use of liposomes, microcapsules ormicrospheres, inclusion complexes, or other types of carriers iscontemplated. Chemically modified pharmaceutical composition of thepresent invention may also be prepared in different formulationsdepending on the type of chemical modification or the type of deviceemployed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise pharmaceutical composition of thepresent invention (or derivative) dissolved in water at a concentrationof about 0.1 to 25 mg of biologically active ingredients of apharmaceutical composition of the present invention per mL of solution.The formulation may also include a buffer and a simple sugar (e.g., forstabilization and regulation of osmotic pressure of a pharmaceuticalcomposition of the present invention). The nebulizer formulation mayalso contain a surfactant, to reduce or prevent surface inducedaggregation of the pharmaceutical composition of the present inventioncaused by atomization of the solution in forming the aerosol.

The liquid aerosol formulations contain a pharmaceutical composition ofthe present invention and a dispersing agent in a physiologicallyacceptable diluent. The dry powder aerosol formulations of the presentinvention consist of a finely divided solid form of a pharmaceuticalcomposition of the present invention and a dispersing agent. With eitherthe liquid or dry powder aerosol formulation, the formulation must beaerosolized. That is, it must be broken down into liquid or solidparticles in order to ensure that the aerosolized dose actually reachesthe mucous membranes of the nasal passages or the lung. The term“aerosol particle” is used herein to describe the liquid or solidparticle suitable for nasal or pulmonary administration, i.e., that willreach the mucous membranes. Other considerations, such as constructionof the delivery device, additional components in the formulation, andparticle characteristics are important. These aspects of nasal orpulmonary administration of a drug are well known in the art, andmanipulation of formulations, aerosolization means and construction of adelivery device require at most routine experimentation by one ofordinary skill in the art.

Often, the aerosolization of a liquid or a dry powder formulation forinhalation into the lung will require a propellent. The propellent maybe any propellant generally used in the art. Specific non-limitingexamples of such useful propellants are a chlorofluorocarbon, ahydrofluorocarbon, a hydrochlorofluorocarbon, or a hydrocarbon,including trifluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof.

Systems of aerosol delivery, such as the pressurized metered doseinhaler and the dry powder inhaler are disclosed in Newman, S. P.,Aerosols and the Lung, Clarke, S. W. and Davia, D. editors, pp. 197-22and can be used in connection with the present invention.

In general, as described in detail infra, pharmaceutical composition ofthe present invention is introduced into the subject in the aerosol formin an amount between about 0.01 mg per kg body weight of the mammal upto about 1 mg per kg body weight of said mammal. In a specificembodiment, the dosage is administered as needed. One of ordinary skillin the art can readily determine a volume or weight of aerosolcorresponding to this dosage based on the concentration ofpharmaceutical composition of the present invention in an aerosolformulation of the invention.

Liquid Aerosol Formulations.

The present invention provides aerosol formulations and dosage forms. Ingeneral such dosage forms contain a pharmaceutical composition of thepresent invention in a pharmaceutically acceptable diluent.Pharmaceutically acceptable diluents include but are not limited tosterile water, saline, buffered saline, dextrose solution, and the like.

The formulation may include a carrier. The carrier is a macromoleculewhich is soluble in the circulatory system and which is physiologicallyacceptable where physiological acceptance means that those of skill inthe art would accept injection of said carrier into a patient as part ofa therapeutic regime. The carrier preferably is relatively stable in thecirculatory system with an acceptable plasma half life for clearance.Such macromolecules include but are not limited to Soya lecithin, oleicacid and sorbitan trioleate, with sorbitan trioleate preferred.

The formulations of the present embodiment may also include other agentsuseful for pH maintenance, solution stabilization, or for the regulationof osmotic pressure.

Aerosol Dry Powder Formulations.

It is also contemplated that the present aerosol formulation can beprepared as a dry powder formulation comprising a finely divided powderform of pharmaceutical composition of the present invention and adispersant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing pharmaceutical composition of thepresent invention (or derivative) and may also include a bulking agent,such as lactose, sorbitol, sucrose, or mannitol in amounts whichfacilitate dispersal of the powder from the device, e.g., 50 to 90% byweight of the formulation. The pharmaceutical composition of the presentinvention (or derivative) should most advantageously be prepared inparticulate form with an average particle size of less than 10 mm (ormicrons), most preferably 0.5 to 5 mm, for most effective delivery tothe distal lung.

In a further aspect, recombinant cells that have been transformed withthe tankyrase gene and that express high levels of the polypeptide canbe transplanted in a subject in need of tankyrase. Preferably autologouscells transformed with tankyrase are transplanted to avoid rejection;alternatively, technology is available to shield non-autologous cellsthat produce soluble factors within a polymer matrix that preventsimmune recognition and rejection.

Methods of Treatment, Methods of Preparing a Medicament.

In yet another aspect of the present invention, methods of treatment andmanufacture of a medicament are provided. Conditions alleviated ormodulated by the administration of the present derivatives are thoseindicated above.

Dosages.

For all of the above molecules, as further studies are conducted,information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, will be able to ascertain proper dosing.

A subject in whom administration of tankyrase is an effectivetherapeutic regiment is preferably a human, but can be any animal. Thus,as can be readily appreciated by one of ordinary skill in the art, themethods and pharmaceutical compositions of the present invention areparticularly suited to administration to any animal, particularly amammal, and including, but by no means limited to, domestic animals,such as feline or canine subjects, farm animals, such as but not limitedto bovine, equine, caprine, ovine, and porcine subjects, wild animals(whether in the wild or in a zoological garden), research animals, suchas mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avianspecies, such as chickens, turkeys, songbirds, etc., i.e., forveterinary medical use.

The present invention may be better understood by reference to thefollowing non-limiting Example, which is provided as exemplary of theinvention. The following example is presented in order to more fullyillustrate the preferred embodiments of the invention. It should in noway be construed, however, as limiting the broad scope of the invention.

EXAMPLE 1 Tankyrase, A PARP-related Enzyme at Human TelomeresIntroduction

Human chromosome ends consist of tandom arrays of telomeric TTAGGGrepeats bound to specific proteins [Bilaud et al., Nature Gen.,17:236-239 (1997); Chong et al., Science, 270:1663-1667 (1995); Broccoliet al., Nature Gen., 17:231-235 (1997)]. Due to the inability ofconventional DNA polymerases to replicate chromosome ends, telomericsequences are lost at each cell division [Cooke and Smith, Cold SpringHarbor Sym. Quant. Biol., LI:213-219 (1986); Harley et al., Nature,345:458-460 (1990); Hastie et al., Nature, 346:866-868; reviewed inHarley, Telomeres and Ageing, In Telomeres (ed. Blackburn and Grieder)Cold Spring Harbor Press, 247-265]. In the germline and in immortalizedcells and tumors, telomeric DNA can be maintained by telomerase, areverse transcriptase that adds TTAGGG repeats onto 3′ ends ofchromosomes [reviewed in Greider, Ann. Rev. Biochem., 65:337-365 (1996);Morin, Seminars in Cell Dev. Biol., 7:5-15 (1996)]. In somatic cells,due to the low level or absence of telomerase, telomeres shorten by50-200 basepairs per cell division. This programmed telomere shorteningmay be best viewed as a tumor suppressor mechanism that limits thegrowth potential of transformed cells [de Lange, Science, 279:333-335(1998)]. In agreement, telomere length is strongly correlated with theproliferative capacity of normal human cells [Allsopp et al., Proc.Natl. Acad of Sci. USA, 89:10114-10118 (1992)], the catalytic subunit oftelomerase (hTERT) is up-regulated in human tumors, and immortalizedcells [Meyerson et al., Cell, 90:785-795 (1997); Nakamura et al.,Science, 277:955-959 (1997)] and activation of telomerase in primaryhuman cells results in the extension of cellular life-span beyond thescheduled senescence point [Bodnar et al., Science, 279:349-352 (1998);Vaziri and Benchimol, Curr. Biol., 8:279-282 (1998)].

The only known protein components of mammalian telomeres are the TRFproteins, duplex TTAGGG repeat binding factors that are localized attelomeres in interphase and metaphase chromosomes [Zhong et al., Mol.Cell. Biol., 13:4834-4943 (1992); Chong et al., Science, 270:1663-1667(1995); Ludérus et al., J. Cell Biol., 135:867-881 (1996); Broccoli etal., Hum. Mol. Genetics, 6:69-76 (1997); see Smith and de Lange, Trendsin Genetics, 13:21-26 (1997) for review]. Human TRF1 (hTRF1) is alow-abundance activity found in nuclear extracts from all human cellsand tissues and a similar activity is present in other vertebrates[Zhong et al., Mol. Cell. Biol., 13:4834-4943 (1992); Chong et al.,Science, 270:1663-1667 (1995)]. TRF2 (also referred to as orf2) wasrecently identified as a TRF1 homolog. [Bilaud et al., Nucl. Acids Res.,24:1294-1303 (1996)]. While the function of the TRFs has not been fullyestablished, similar duplex telomeric DNA binding activities in yeastshave been implicated in telomere length control, telomere stability, andtelomeric silencing [reviewed in Shore, Trends Gen., 10:408-412 (1994);Zakian, Saccharomyces telomere: function, structure and replication,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 107-138(1995a); see also McEachern and Blackburn, Nature, 376:403-409 (1995);Krauskopf and Blackburn, Nature, 383:354-357 (1996)].

TRF1 has DNA binding properties in vitro that are consistent with itspresence along the double-stranded telomeric repeat array at chromosomeends. TRF1 binds efficiently to arrays of duplex TTAGGG repeats,irrespective of the presence of a DNA terminus [Zhong et al., Mol. Cell.Biol., 13:4834-4943 (1992)]. Single-stranded telomeric DNA is not aneffective TRF1 substrate and neither are heterologous telomericsequences, such as double-stranded arrays of TTGGGG, TTAGGC, TTTAGGG,TTAGGGGG, and TAGGG repeats [Zhong et al., Mol. Cell. Biol.,13:4834-4943 (1992); Hanish et al. Proc. Natl. Acad. Sci. USA,91:8861-8865 (1994); Chong et al., Science, 270:1663-1667 (1995)]. Thissequence specificity of TRF1 matches the sequence requirements for denovo telomere formation in human cells, suggesting that the TRF proteinsare involved in this process [Hanish et al. Proc. Natl. Acad. Sci. USA,91:8861-8865 (1994)].

A novel human telomeric protein, tankyrase, has been isolated, asdescribed herein, that binds TRF1 and is located at human telomeresthroughout the cell cycle. Tankyrase was isolated using a two-hybridscreen with TRF1 on the premise that telomere length homeostasisinvolves additional TRF1- and telomerase-associated proteins. The domainstructure of tankyrase indicates a mechanism by which TRF1 mightregulate telomerase.

Methods

Tankyrase cDNA Cloning:

The full-length tankyrase cDNA TT20 contains a 4134 nucleotide (nt)insert in the vector pBKCMV. It has an ORF of 1327 amino acids startingwith CGAAGATGG initiating codon (6 nt in from the 5′ end), which isfavorable for initiating translation. Two other overlapping isolates TT6and TT18, which contained 23 nt 5′ of the end of TT20, had an in-framestop codon upstream of the initiating ATG, confirming the translationalstart site. The 3′ end of TT20 contained a stop codon followed by 146 ntof 3′ untranslated sequence.

The TT20 cDNA was isolated in several steps. First, a PCR product(encoding amino acids 973-1163 of SEQ ID NO:2) made from TR1L-4 was usedas a probe to screen a HeLa cell cDNA library. Two overlapping cDNAs, 32and 21, encompassing 8,901 nt were isolated. These clones encoded aminoacids 235-1327 of SEQ ID NO:2. The 3′ end had 5,539 nt of 3′untranslated sequence and a AATAAA polyadenylation site 19 nt upstreamof a poly A stretch. The 5′ end sequence was extended using the RACEprocedure to yield a 514 nt clone RACE 4C (encoding amino acids 83-253of SEQ ID NO:2). A continuous open reading frame (ORF) was constructed(RACE4C+32) and a PCR probe derived from this construct, encoding aminoacids 183-303 of SEQ ID NO:2, was used to screen a human testis library(Stratagene) to isolate TT20 as described above.

Two other testis library isolates and TT9 were characterized. DNAsequence analysis indicated that they had the same 5′ end as TT20.Restriction digest and nested PCR analysis indicated they were similarto TT20 along their length except each had an approximately 100 ntinsertion; TT7, had an insertion after amino acid 640 of SEQ ID NO:2 (inANK repeat 14) and TT9, insertion after amino acid 881 of SEQ ID NO:2(in ANK repeat 21). Both insertions contained stop codons resulting intruncated proteins which were confirmed by in vitro translation.

Tankyrase Expression Constructs:

FLAG-tankyrase-1 (encoding amino acids 337-1149 of SEQ ID NO:2) wasconstructed by cloning a PCR amplified fragment into the NotI-ApaIcloning sites of a modified pRc/CMV expression vector (Invitrogen)carrying a FLAG epitope 5′ of the cloning sites. PCR was performed onplasmid TR1L-4 as template with 5′ TTGCGGCCGCAGACGAACTCCTAGAAGCT 3′ asforward primer and 5′ GCGGGCCCTATCGAATGACATTGTATCTGT 3′ as backwardprimer. FLAG-tankyrase (encoding amino acids 2-1327 of SEQ ID NO:2) wasconstructed in two steps. First, an intermediate construct CMV-IMC(encoding amino acids 2-182 SEQ ID NO:2) was made by cloning aPCR-amplified fragment into the NotI-ApaI cloning sites of the modifiedpRc/CMV vector described above. PCR was performed on plasmid TT20 astemplate with 5′ TTGCGGCCGCGGCGGCGTCGCGTCGCT 3′ as forward primer and 5′TGCGGCGTCCACCACGGT 3′ as backward primer. The subsequent digestion cutthe natural ApaI site at amino acid 182 of SEQ ID NO:2. Next an ApaIfragment (encoding amino acids 183-1327 of SEQ ID NO:2, a stop codon,146 nt of 3′ untranslated sequence, and vector polylinker sequence) fromTT20 was cloned into the ApaI site of CMV-IMC and screened for thecorrect orientation to yield FLAG-tankyrase.

Yeast Two-hybrid Analysis:

TR1L-4 and TR1L-12 were isolated from a human liver two-hybrid cDNAlibrary (Clontech) created in pGad10. The library was screened withhuman full length TRF1 cDNA fused to LexA (LexA-TRF1) [Bianchi et al.,EMBO J., 16:1785-1794 (1997)] in the yeast strain L40 as described[Hollenberg et al., Mol. Cell Biol., 15:3813-3822 (1995)]. Two-hybridanalysis was performed as described by Bianchi et al. [EMBO J.,16:1785-1794 (1997)]. β-galactosidase assays for the two-hybrid analysiswas performed as described by Bianchi et al. [EMBO J., 16:1785-1794(1997)].

Anti-tankyrase Antibodies:

The Ank2 plasmid containing a sub-domain of tankyrase (encoding aminoacids 973-1149 of SEQ ID NO:2) in the vector pET-22b(+) (Novagen) wasexpressed as a fusion protein in E. coli. The protein was isolated ininclusion bodies and used to immunize a rabbit (#465). The resultingimmune serum, rabbit anti-tankyrase, 465, was affinity purified againstAnk2 protein coupled to CnBr-activated SEPHAROSE (Sigma Biochemicals)using standard procedures [Harlow and Lane. Antibodies, A LaboratoryManual, Cold Spring Harbor Press, (1988)].

PARP assays were performed with baculovirus-derived tankyraseessentially as described in [Simonin et al. 268:8529 (1993)] but withoutaddition of DNA. To make baculovirus-derived protein, an N-terminally[His]₆-tagged fusion protein of human tankyrase was generated in theexpression vector pFastBac HTb (Gibco BRL, Grand Island) and used togenerate a recombinant plasmid in DH10Bac E. coli. The recombinant DNAwas used to transfect SF21 insect cells and recombinant virus wasisolated and amplified. Protein was purified as described forbaculovirus-derived TRF1 [Bianchi et al., EMBO J. 16:1785 (1997)].Samples containing tankyrase (0-4 μg) and TRF1 (0-4 μg) [Bianchi et al.,EMBO J. 16:1785 (1997)] were incubated for 30 minutes at 25° C. in 0.1ml of assay buffer containing 50 mM Tris-HCl (pH 8.0), 4 mM Mg Cl₂, 0.2mM dithiothreitol (DTT), 1.3 μM [³²P]NAD+ (4 μCi) and varyingconcentrations of unlabeled NAD+ (0-1 mM). Reactions were stopped by theaddition of 20% trichloroacetic acid (TCA). Acid-insoluble proteins werecollected by centrifugation, rinsed in 5% TCA, suspended in Laemmliloading buffer, and fractionated on SDS-PAGE. Proteins were visualizedby Coomassie-Blue stain and autoradiography. For the immunoblot analysisreactions were performed the same way except that the [³²P]NAD⁺ wasomitted. Samples were immunoblotted as described below, and probed with10H, a mouse monoclonal antibody raised against poly(ADP-ribose) (1:250)[Kawamitsu et al., Biochemistry 23:3771 (1984)] followed by horseradishperoxidase-conjugated sheep antibody to mouse IgG (Amersham).

Northern Blot:

Northern blots (Clontech) were probed with the tankyrase cDNA isolatedfrom TR1L-4 as described [Chong et al., Science, 270:1663-1667 (1995)].

Cell Extracts and Protein Fractions:

HeLaI cells were suspended directly in Laemmli loading buffer. For rattestis extracts, crude nuclei were isolated (after hypotonic lysis),extracted with 0.4 M KCl , pelleted and suspended in Laemmli buffer[Chong et al., Science, 270:1663 -1667 (1995)]. Rat nuclei were preparedas described [Blobel and Potter, Science, 154:1662 -1665 (1966)].Nuclear envelopes were prepared according to [Mutanis et al., J. Cell.Biol., 135:1451-1470 (1996)]. Salt-washed nuclear envelopes, prepared asin [Snow et al., J. Cell Biol., 104:1143-1156 (1987)] were extractedwith urea as described [Worman, Proc. Natl. Acad. Sci. USA, 85:8531-8534(1988)].

Western and Northern Blotting:

Proteins samples were fractionated on SDS polyacrylamide gels,transferred to nitrocellulose electrophoretically, and blocked in 5%milk in PBS containing 0.1% Tween-20. Antibody incubations were in 1%milk in PBS containing 0.1% Tween-20. Blots were incubated with affinitypurified rabbit anti-tankyrase (4 μg/ml), pre-immune serum from theanti-tankyrase rabbit (1:500) or affinity purified rabbit anti-TRF 1620.1 (1:50), followed by horseradish peroxidase-conjugated donkeyanti-rabbit IgG (1:2,500). Bound antibody was detected using theenhanced chemiluminescence kit (Amersham). Northern blots (Clontech)were probed with the tankyrase cDNA isolated from TRIL-4 as described inBroccoli et al. [Mol. Cell Biol., 16:3765-3772 (1996)].

For immunoblots, HeLa cells were suspended directly in Laemmli loadingbuffer and loaded at ˜50,000 cells per lane. Crude nuclei were isolatedfrom rat testis (after hypotonic lysis), extracted with 0.4 M KCl,pelleted and suspended in Laemmli buffer. In vitro translated tankyrasewas generated using a coupled transcription/translation reticulocytelysate system (from Promega). One μg of TT20 was incubated with T3 RNApolymerase under standard conditions and 10% of the reaction was loadedper lane. Protein samples were fractionated on SDS polyacrylamide gels,transferred to nitrocellulose, and blocked in 5% milk inphosphate-buffered saline (PBS) containing 0.1% Tween-20. Antibodyincubations were in 1% Tween-20. Blots were first incubated with rabbitanti-tankyrase antibodies (4 μg/ml) or rabbit pre-immune serum (1:500)and then with horseradish peroxidase-conjugated donkey antibody torabbit immunoglobulin G (IgG), (1:2,500), (from Amersham). Boundantibody was detected by enhanced chemiluminescence (Amersham).

For Immunoprecipitation analysis, 80 μg of tankyrase in 1 ml of buffer D[20 mM Hepes (pH 7.9) containing 100 mM KCl, 20% glycerol, 0.2 mMethylenediaminetetraacetic acid (EDTA), 0.2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM DTT, 0.5 mMphenylmethylsulfonyl fluoride (PMSF), 0.1% NP40, 0.1% Triton X-100, and1 mg BSA per ml] was precleared by incubation with an irrelevant rabbitserum at room temperature for 1 hour, followed by addition of protein GSEPHAROSE (Pharmacia). Non-specific antibody complexes and proteinaggregates were removed by centrifugation and the supernatant was usedfor immunoprecipitation analysis. 0.5 ml of supernatant was incubatedwith 2 μg anti-tankyrase antibody or 2 μg preimmune IgG from the samerabbit [purified by affinity chromatography on protein G SEPHAROSE(Pharmacia)] for 1 hour at room temperature. Antigen-antibody complexeswere collected on protein G beads, washed 3 times with buffer D and 2times with 50 mM Tris-HCl pH 8.0. The beads were then assayed for PARPactivity by addition of 20 μl containing 50 mM Tris-HCl (pH 8.0), 4 mMMgCl₂, 0.2 mM DTT, and 1.3 μM [³² P]NAD⁺ (0.8 μCi). The reactions wereincubated and processed as described above.

Gel-shift assays were performed using an end-labeled 142 bpHindlll-Asp718 fragment from plasmid pTH12 [Z. Zhong, et al, Mol. Cell.Biol. 12:4834 (1992)] containing 12 tandem TTAGGG repeats.Baculovirus-derived TRF1 (13 to 120 ng) [Bianchi et al. EMBO J. 16:1785(1997)] was incubated for 30 minutes at room temperature in a 20 μlreaction containing 20 mM Hepes-KOH (pH 7.9), 100 mM KCL, 0.5 mM DTT, 5%glycerol, 0.1% NP40, 100 ng sheared E. coli DNA, 100 ng β-casein, and 1ng of labeled probe. In some cases, reactions were supplemented withNAD⁺ (0.2 mM) and baculovirus-derived human tankyrase (2.5 to 200 ng).Samples were fractionated on a 0.7% agarose gel run in 0.1× TBE (8.9 mMTris-base, 8.9 mM Boric acid, and 0.2 mM EDTA) at 130 volts for 1 hourat room temperature. Gels were dried onto Whatman DE81 paper andautoradiographed.

Tankyrase protein was also detected by Western analysis in the followinghuman cell lines: 293, transformed embryonic kidney cells, IMR90 andWI38, primary lung fibroblasts; WI38 VA13/2RA, immortalized lungfibroblasts; GM847, SV40 immortalized fibroblasts; Daudi and Raji,lymphoma; HT1080, fibrosarcoma; and MCF, breast adenocarcinoma. Severalof these cell lines were found to express only the larger set oftankyrase mRNAs (6-10 kilobases) indicating that the 142 kD polypeptidecan be expressed from one of these transcripts.

Transfection:

HelaI cells were transfected by electroporation of FLAG-tankyrase orFLAG-tankyrase-1 and pcDNA3-hTRF1 cloned into the expression vectorpcDNA3 (Invitrogen). Cells were grown for 16 hr and then processed forimmunofluorescence or immunoprecipitation as described below.

Indirect Immunofluorescence:

HelaI or Hela1.2.11 cells, a subclone of HeLaI containing telomeres ofmore than 20 kb, were fixed with ice cold methanol at −20° C. for 10 minor 3.7% formaldehyde in PBS for 10 min followed by permeabilization with0.5% NP40 in PBS for 10 min. For chromosome spreads, Hela1.2.11 cellswere treated with colcemide (0.1 μg/ml, 60 min), harvested bytrypsinization, hypotonically swollen in 10 mM Tris (pH7.4), 10 mM NaCland 5 mM MgCl₂ and sedimented onto coverslips for 15 seconds at 3000 rpmin a Sorvall RT6000B tabletop centrifuge. Chromosomes were swollen for15 min in 25% PBS, then fixed in 3.7% formaldehyde in 25% PBS for 10min, followed by permeabilization with 0.5% NP40 in 25% PBS for 10 min.Samples were blocked with 1%BSA in PBS, followed by incubation withprimary antibodies diluted in 1% BSA/PBS. Endogenous tankyrase wasdetected with affinity-purified rabbit anti-tankyrase 465 (1-4 μg/ml).FLAG-tankyrase was detected with the mouse monoclonal antibody M2anti-FLAG (Eastman-Kodak) (2-10 μg/ml). Nuclear pore complex proteinswere detected with a mouse monoclonal antibody 414 [Davis and Blobel,Cell, 45:699-709 (1986)] (supernatant, 1:100). Centrosomal proteins inuntransfected cells were detected with mouse monoclonal antibodies to:NuMA1F1 [Compton et al., J. Cell Biol., 112:1083-1097 (1991)], (ascites1:100), centrin 20H5 [Sanders and Salisbury, J. Cell Biol., 124:795-805(1994)] (ascites 1:2000), and γ-tubulin (ascites 1:2000) (Sigma). Intransfected cells γ-tubulin was detected with a rabbit anti-peptideantibody XGC-1-4 (1:2000). Endogenous TRF1 in Hela1.2.11 cells wasdetected with mouse polyclonal serum directed against full length TRF1(1:10,000). TRF1 in HeLa cells was detected with rabbit anti-TRF1antibody 371 [van Steensel and de Lange, Nature, 385:740-473 (1997)](0.4 μg/ml) for untransfected cells and (0.04 μg/ml) for transfectedcells. Primary antibodies were detected with FITC- or TRITC-conjugateddonkey anti-mouse or rabbit antibodies (1:100) (Jackson Laboratories).DNA was stained with DAPI (0.2 μg/ml). Micrographs were recorded on aZeiss Axioplan microscope with a Photometric CCD camera. Images wereprocessed and merged using Adobe Photoshop. Immunolocalization analysisof cycling HeLa cells indicates additional subcellular locations fortankyrase.

Immunoprecipitation:

Whole cell extracts were prepared from transfected HeLaI cells asdescribed [van Steensel et al., Cell, 92:401-413 (1998)]. Proteins wereimmunoprecipitated overnight on ice by addition of anti-tankyraseantibodies 465 (1 μg/ml), anti-TRF1 antibody 371 (0.1 μg/ml) or anunrelated rabbit serum as a control. Antibody antigen complexes werecollected on protein G beads and processed as described [Broccoli etal., Nature Gen., 17:231-235 (1997)].

Immunoelectron Microscopy:

HelaI cells in tissue culture dishes were permeabilized for 15 secondsin 0.5% Triton×-100/PBS, washed 2× in PBS, fixed for 10 min in 3%formaldehyde/PBS and blocked in 1% BSA/PBS. Cells were incubated withaffinity purified rabbit anti-tankyrase antibodies 465 (5 μg/ml),followed by 5 nm gold-conjugated anti rabbit antibodies. Samples wereprocessed for thin sectioning and electron microscopic analysis asdescribed [Pain et al., Nature, 347:444-449 (1990)].

Amino Acid Alignments:

Alignment of the 24 ANK repeats is based upon a Megalign Clustalalignment (gap penalty 10, gap length penalty 10) of the tankyrase ANKrepeat domain with the ANK repeat domains of human ankyrins 1 (Genbank#M28880), 2 (Genbank #X56958) and 3 (Genbank #U13616). Comparisons ofthe PARP-related and SAM domains of tankyrase with other proteins wasdone with Clustal W 1.6 (gap opening penalty 10, gap extension penalty0.05).

Results

Isolation of Tankyrase cDNA and Analysis of its Predicted PrimaryStructure:

A yeast two-hybrid screen with human TRF1 as bait was performed. Uponscreening 1×10⁷ transformants of a human fetal liver two-hybrid library,13 positives were obtained. 12 of these contained an identical 2.4 kbinsert, designated TR1L-4 and one had a 1 kb insert, designated TR1L-12,which was contained within TR1L-4 (see FIG. 1A). DNA sequence analysisindicated that TR1L-4 was a partial cDNA. Conceptual translation of thecDNA revealed that it was a novel protein, although it contained 20copies of the previously recognized ANK repeat motif (see below). A fulllength cDNA (SEQ ID NO:1), designated tankyrase (TRF1-interactingankyrin), isolated from a human testis library, contained an openreading frame of 1327 amino acids (SEQ ID NO:2), predicted to encode aprotein of 142 kD.

A schematic representation of the predicted primary structure oftankyrase is presented in FIG. 1A. The amino terminal HPS domainconsists of homohistidine, proline and serine tracts. Proline richsequences have been shown to serve as binding sites for SH3 domains. Astriking feature is the central domain containing 24 ANK repeats, a 33amino acid motif shown to mediate protein-protein interactions [Bork,Proteins, 17:363-374 (1993); Michaely and Bennett, Trends Cell Biol.,2:127-129 (1992)]. ANK repeats are found in multiple copies, typically 4to 8, in a functionally diverse group of proteins that includes theankyrins, a family of structural proteins that link integral membraneproteins to the underlying cytoskeleton [reviewed in Bennett, J. Biol.Chem., 267:8703-8706 (1992)]. Ankryins are notable for containing anunusually high number (24) of ANK repeats.

Several observations suggest that tankyrase is not just an ANK-repeatcontaining protein, but rather, a new member of the ankyrin family.First, the ANK repeats in tankyrase and the ankryins sharescharacteristic features that distinguishes them from the ANK repeatsfound in other proteins, such as the presence of a hydrophobic aminoacid at position 3 and an N or D at position 29 (FIG. 1B) [Peters andLux, Semin. Hematol., 30:85-118 (1993)]. Second, ankyrins consist of 24(mostly perfect) 33 amino acid repeats with the exception of repeat 5,which is 29 amino acids. While tankyrase consists of more irregularrepeats, its shortest repeat is also repeat 5, which is 25 amino acids.Overall, the repeat domains of the ankyrins are 32-39% identical withthe 830-amino acid repeat domain of tankyrase. Together, theseobservations indicate that tankyrase is related to the ankyrin familyand as such may play a structural role in the cell. Apart from the ANKrepeat domain, however, there was no detectable homology betweentankyrase and ankyrins.

The carboxy terminal domain of tankyrase contained another motifpostulated to function in protein-protein interaction. SAM (sterilealpha motif) is a 65-70 amino acid domain found in 1-3 copies in adiverse group of proteins implicated in developmental processes[Ponting, Protein Science, 4:1928-1930 (1995); Schultz et al., ProteinScience, 6:249-253 (1997)]. An alignment of this motif with threeunrelated SAM-containing proteins is presented in FIG. 1C. Two types ofinteractions have been shown for SAM domains; homo- or heterotypicinteraction with other SAM domains [Barr et al., Mol. Cell Biol.,16:5597-5603 (1996)] or binding to SH2 domains via phosphorylation of aconserved tyrosine [Stein et al., J. Biol. Chem., 271:23588-23593(1996)]. Since the SAM domain of tankyrase does not contain theconserved tyrosine required for binding SH2 domains, its binding partneris likely to be another SAM domain.

Finally, a 150 amino acid domain in the carboxyl terminus of tankyraseshowed homology to poly(ADP-ribose) polymerase (PARP), a highlyconserved nuclear protein, found in most eukaryotes except S. cerevisiae[for review see: de Murcia and de Murcia, Trends Biochemical Sciences,19:172-176 (1994); Jeggo, Curr. Biol., 8:R49-51 (1998); Lindahl et al.,Trends in Biochemical Sciences, 20:405-411 (1995)]. In response to DNAdamage, PARP catalyses the formation of poly(ADP-ribose) onto a proteinacceptor using NAD⁺ as a substrate. The homology falls in the catalyticdomain of PARP, which binds NAD⁺. Structural analysis indicated thatthis domain consisted of secondary structure units (multiple β strandsand one alpha helix; indicated in FIG. 1D) [Ruf et al., Proc. Natl.Acad. Sci. USA, 93:7481-7485 (1996)], that form a cavity known as theNAD⁺-binding fold, a tertiary structure that is also present in allADP-ribosylating toxins [Domenighini et al., Mol. Microbiol., 14:41-50(1994)]. PARP and the toxins constitute a superfamily ofADP-ribosyl-transferases [Ruf et al., Proc. Natil. Acad. Sci. USA,93:7481-7485 (1996)]. The identities between tankyrase and PARP fallwithin these secondary structure units. FIG. 1D shows an alignment ofhuman and Drosophila (derived from a 508 nt sequence in the ESTdatabase) tankyrase, human and Drosophila PARP, and an uncharacterizedhuman cDNA in the database (KIA0077), which, like tankyrase, ishomologous to PARP only in this domain. Several features are worthnoting, First, all the amino acids in PARP that have been implicated inNAD⁺-binding or catalysis are conserved in tankyrase, including criticalamino acids that are conserved between the eukaryotic PARPs and theprokarytic ADP-ribosylating toxins, DT (dimeric diphtheria toxin) andETA (exotoxin A from Pseudomonas aeruginosa [see Ruf et al., Proc. Natl.Acad. Sci. USA, 93:7481-7485 (1996)]). Second, human and Drosophilatankyrase are even more conserved (80% identical) in this region thanhuman and Drosophila PARP (65% identical), highlighting the importanceof this domain in tankyrase. Third, the three human proteins (tankyrase,PARP and KIA0077) share 28-30% identity in this domain. Here again, allthe critical residues are conserved and most of the identical residuesfall within the secondary structure units that form the NAD⁺-bindingfold. Thus, based upon these observations it is concluded that tankyraseis a new member of the ADP-ribosyl-transferase superfamily of enzymeswith PARP as its closest relative. The conservation of the amino acidscritical for NAD⁺-binding and catalysis suggests that tankyrase encodesa similar enzymatic activity.

Tankyrase is Ubiquitously Expressed:

The expression pattern of tankyrase was evaluated by Northern blotanalysis of RNA from a variety of human tissues (FIG. 2A). The tankyrasecDNA hybridized to three mRNAs of ^(˜)6, 8, and 10 kb with the sameubiquitous expression pattern as TRF1 and TRF2 [Broccoli et al., NatureGen., 17:231-235 (1997); Chong et al., Science, 270:1663-1667 (1995)].The tankyrase message was particularly abundant in testis where therewere two additional messages of ^(˜)2.5 and 4.5 kb. The 4.2 kb tankyrasecDNA isolated from the testis library is large enough to represent theabundant 4.5 kb transcript suggesting that this cDNA is nearlyfull-length. The larger transcripts present in most tissues may be dueto longer 3′ untranslated regions. Indeed, DNA sequence analysis of atankyrase cDNA isolated from a HeLa cell library revealed the samesequence as the full-length 4.5 kb cDNA but, had an additional 5 kb of3′ untranslated sequence. When the most 3′ 1 kb of this cDNA was used asa probe in a Northern blot, it hybridized exclusively to the largest (10kb) transcript supporting the idea that the larger transcripts reflectadditional 3′ untranslated regions (UTRs).

To analyze the expression of the tankyrase protein, polyclonalantibodies were raised against a subdomain of tankyrase (indicated asAnk2, see FIG. 1A), expressed as a fusion protein in E. coli. Immunoblotanalysis with affinity purified anti-ankyrin antibodies (FIG. 2B)revealed a single polypeptide of ^(˜)142 kD (the predicted molecularweight) in rat testis cell extracts (FIG. 2B, lane 1) and human HeLaIwhole cell lysates (FIG. 2B, lane 2). The protein co-migrated withimmunoreactive, in vitro translated tankyrase (FIG. 2B, lane 3),indicating that the cDNA encoded the full-length protein. Consistentwith the idea that the multiple tankyrase transcripts differed only intheir 3′ untranslated regions, only a single immunoreactive polypeptidewas expressed despite the complex pattern of transcripts (particularlyin testis). The specificity of the antibody was confirmed by the lack ofreactivity with preimmune serum (FIG. 2B, lanes 4-6).

Localization of Exogenous Tankyrase to Telomeres is TRF1-dependent:

Initially the tankyrase cDNA was used to determine the subcellularlocalization of the protein. A construct was prepared containing thefull-length tankyrase cDNA which also encoded a FLAG epitope at theN-terminus of tankyrase. The construct was expressed by transienttransfection in HeLaI cells. Indirect immunofluorescence withanti-FLAG-antibodies indicated a cytoplasmic staining pattern for thetransfected protein (FIG. 3A). Co-staining with TRF1 antibody (FIG. 3B)showed that tankyrase did not co-localize with TRF1 and, in fact, wasexcluded from the nucleus (FIG. 3C). When tankyrase was co-transfectedwith TRF1 it displayed a different pattern of localization;FLAG-tankyrase was translocated from the cytoplasm to the nucleus (FIG.3E) where it co-localized with TRF1 in a punctate pattern (FIG. 3G)consistent with a telomeric localization. Similarly, in co-transfectedmitotic cells FLAG-tankyrase co-localized with TRF1 in a patternconsistent with localization to telomeres (FIG. 3K). A telomericstaining pattern for tankyrase was only observed in cells overexpressingTRF1. Note that in these experiments the anti-TRF 1 antibodies did notdistinguish between exogenous and endogenous TRF1. However,TRF1-transfected cells were easily recognized by the increased level ofTRF1 expression. These findings confirmed the two hybrid result byshowing that TRF1 and tankyrase interacted in mammalian cells and alsosuggested that transport of tankyrase into the nucleus to telomeres waslinked to TRF1 synthesis (see below).

To determine if tankyrase and TRF1 were actually physically complexed incells, co-immunoprecipitation experiments were performed on transfectedcell extracts. Due to a low efficiency of transient expression withtransfection of full-length tankyrase, these studies were done with amore effective expression plasmid, tankyrase-1, containing a partialtankyrase ORF, (TR1L-4, see FIG. 1A) with a FLAG epitope at its aminoterminus. Indirect immunofluorescence with anti-FLAG antibody indicatedthat transfected tankyrase-1, like full-length tankyrase, localized totelomeres when co-transfected with TRF1. Extracts prepared fromtankyrase-1/TRF1 transfected cells were subjected to immunoprecipitationanalysis followed by immunoblotting. As shown in FIG. 4A, TRF1 wasimmunoprecipitated with anti-tankyrase antibodies (FIG. 4A, lane 2), andconversely, transfected tankyrase-1 was immunoprecipitated withanti-TRF1 antibodies (FIG. 4A, lane 5), demonstrating that the proteinswere complexed in vivo. Only a small fraction of the total protein ineach case was co-immunoprecipitated, consistent with the stainingpattern in co-transfected cells (see for example FIG. 3E).

Two-hybrid analysis was used to determine the interacting domainsbetween tankyrase and TRF1. The smallest isolate from the originaltwo-hybrid screen, TR1L-12 (see FIG. 1A), consisted of only 10 internalANK repeats (ANK repeats 9-19), thereby demonstrating that tankyraseinteracted with TRF1 through its ANK repeats. To determine thetankyrase-interacting domain in TRF1, two-hybrid analysis was performedwith the 10-ANK repeat domain of TR1L-12 fused to the GAL4 activationdomain (GAD-tankyrase) and full-length and deletion constructs of TRF1fused to LexA. As shown in FIG. 4B co-expression of full-length TRF1fused to LexA (LexA-FL) and GAD-tankyrase resulted in transcriptionalactivation of the lacZ reporter gene that was dependent upon both TRF1and tankyrase sequences. As observed previously, the amino-terminalacidic domain of TFR1 (LexAd68-C) activated transcription even in theabsence of tankyrase sequences in the GAD fusion partner. However, thisactivity increased significantly from 12.0 to 50.4 units when the GADfusion partner contained tankyrase sequences. Deletion of the acidicdomain of TRF1 (ΔN66-LexA) abolished the interaction with GAD-tankyrase.Together, these results demonstrated that the amino-terminal acidicdomain of TRF1 is necessary and sufficient for interaction with the10-ANK repeat domain of tankyrase.

Tankyrase is Located at Nuclear Pore Complexes in Interphase and atCentrosomes in Mitosis:

Next the subcellular localization of endogenous tankyrase wasdetermined. Indirect immunofluorescence of HeLaI cells with affinitypurified anti-tankyrase antibody indicated that tankyrase localized tothe nuclear envelope in interphase and to the centrosomes in mitosis(FIG. 5A, panel 1). This staining pattern was blocked if the antibodieswere preincubated with the recombinant tankyrase fusion protein (Ank2,see FIG. 1A), against which the antibody was raised, prior toimmunfluorescence. The punctate nuclear rim stain was reminiscent ofnuclear pore complex stain. Indeed co-staining of cells with MAb414, amonoclonal antibody that recognizes a family of nuclear pore complexproteins [Davis and Blobel, Cell, 45:699-709 (1986)], revealed anidentical staining pattern at the nuclear rim, but not at centrosomes(FIG. 5A, compare panels 1 and 2).

The nuclear envelope localization of tankyrase was confirmed byimmunoblot analysis of subcellular fractions of rat liver (FIG. 5B).Tankyrase was highly enriched in the nuclear envelope fraction (FIG. 5B,lane 5) and remained bound even after extraction with 0.5 M NaCl and 8 Murea (FIG. 5B, lane 8), indicating a tight association with nuclearenvelopes. Resistance to extraction by 8 M urea (which removes tightlyassociated, peripheral membrane proteins including the nuclear lamins;see FIG. 5B, top panel, lane 7) is usually a property of integralmembrane proteins. However, tankyrase is unlikely to be an integralmembrane protein since its predicted amino acid sequence does notindicate a strong transmembrane domain since it does not associate withmicrosomal membranes when co-translated in vitro. The tight associationbetween tankyrase and nuclear envelopes reflects an unusual property ofthe ANK repeat domain.

To further characterize the nuclear envelope localization, immunogoldelectron microscopy with affinity purified anti-tankyrase antibodies wasperformed. As expected, (from the cell staining and fractionation),tankyrase localized to the nuclear envelope, specifically to thecytoplasmic face of the nuclear pore complex (FIG. 5C). Tankyrase oftenappeared to be located on the tips of the fibers that emanate from thenuclear pore complex into the cytoplasm. In addition to the predominantcytoplasmic location, occasionally one or two gold particles appeared onthe nuclear face of the nuclear pore complex. The low level of signal onthe nuclear side of the nuclear pore complex could be due to aninaccessibility of tankyrase to the antibody. Immunogold labeling oftankyrase at the nuclear pore complex required pretreatment of cellswith Triton X-100 prior to fixation, suggesting that tankyrase epitopesare inaccessible. Nonetheless, under the conditions used, tankyraselocalizes predominantly to the cytopolasmic face of the nuclear porecomplex and a minor fraction, possibly more, to the nuclear side.

As shown in FIG. 5A tankyrase localized to the centrosome in mitosis.Tankyrase first appeared at the centrosome in early prophase andremained there throughout mitosis to telophase, reaching maximalaccumulation at metaphase (see FIG. 5A, panel 1). The centrosomallocation of tankyrase was further investigated by a series ofdouble-label immunofluorescence experiments with antibodies directedagainst previously characterized centrosomal proteins, includingcentrin, a component of the centrioles [reviewed in Salisbury, Curr.Opin. Cell Biol., 7:39-45 (1995)], γ-tubulin, a pericentriolar matrixprotein [Stearns et al., Cell, 65:825-836 (1991); Zheng et al., Cell,65:817-823 (1991)] and NuMA, which accumulates around the pericentriolarmatrix protein upon nuclear envelope breakdown [reviewed in Cleveland,Trends Cell Biol., 5:60-64 (1995)]. As shown in FIG. 6, tankyrase didnot co-localize with centrin (FIG. 6C), or γ-tubulin (FIG. 6F),indicating that tankyrase is not an integral component of the centrosomeper se. However, tankyrase did co-localize with NuMA (FIG. 6I) aroundthe pericentriolar matrix region. To confirm the centrosomal location oftankyrase by a means other than the anti-tankyrase antibodies, thedistribution of exogenous FLAG-tankyrase at mitosis was determined. Asshown in FIGS. 6J and 6L, FLAG-tankyrase localized to the centrosome,around the pericentriolar matrix region, similar to endogenous tankyrase(as shown by γ-tubulin staining, FIG. 6K).

Tankyrase is Located at Telomeres Throughout the Cell Cycle:

The absence of endogenous tankyrase at telomeres was surprising sinceexogenous tankyrase interacted with TRF1 and co-localized with TRF1 in atelomeric staining pattern. The possibility therefore existed that theamount of tankyrase at telomeres was below the level of detection andtherefore, cells with longer telomeres might allow detection oftelomeric tankyrase. To address this, indirect immunofluorescence wasperformed on HeLaI.2.11 cells, a clonal isolate of HeLal cells with longtelomeres (greater than 20 kb). For these experiments cells were fixedwith methanol which eliminates the nuclear envelope staining patternobserved with formaldehyde-fixed cells (see FIG. 5A, panel 1). As shownin FIG. 7A, staining of methanol-fixed HeLaI.2.11 cells withanti-tankyrase antibodies revealed a nuclear punctate pattern ininterphase cells which coincided with TRF1 staining (FIG. 7C),indicating a telomeric location for tankyrase in interphase. Unlike thepattern seen in formaldehyde-fixed cells (FIG. 5A, panel 1), themethanol fixation also revealed a residual cytoplasmic localization fortankyrase (FIG. 7A). To determine if tankyrase localized to chromosomesduring mitosis, indirect immunofluorescence was performed on metaphasespreads from HeLaI.2.11 cells. In order to detect tankyrase, metaphasespreads were first swollen in hypotonic buffer, followed by formaldehydefixation in hypotonic buffer. As shown in FIG. 7D, tankyrase wasdetected predominantly at chromosome ends. Most metaphase chromosomeshad tankyrase at their ends where it colocalized with TRF1 (FIG. 7F).Occasionally, telomeres were observed without tankyrase, but thisappeared to be a random occurrence and most likely reflected difficultyin detection. These results demonstrate that tankyrase localizes totelomeres in vivo throughout the cell cycle.

To investigate whether tankyrase has PARP activity, baculovirus-derivedrecombinant protein was tested in an assay that measures the addition ofradiolabeled ADP-ribose to protein acceptors using [³²P]NAD⁺ as asubstrate (see Methods, above). Incubation of tankyrase in the presenceof 1.3 μM radiolabeled NAD⁺ produced ³²P-labeled species thatco-migrated with tankyrase, suggested that tankyrase has the abilityADP-ribosylates itself (FIG. 9A). Higher concentrations of NAD⁺ (0.04 to1 mM) yielded much larger products, likely reflecting the addition ofpoly(ADP-ribose) to tankyrase. The generation of ADP-ribosylatestankyrase depended on the concentration of tankyrase (FIG. 9A), and waseliminated by heat-inactivation of the enzyme. The ADP-ribosylatingactivity could also be removed by immunoprecipitation withanti-tankyrase antibody (FIG. 9B, and see Methods). These resultsindicate that the PARP activity is an intrinsic property of tankyrase.

Tankyrase also has the ability to modify TRF1. At low NAD⁺ concentration(1.3 μM) the ADP-ribosylates products co-migrated with TRF1, whereas athigher NAD⁺ concentrations (0.04 to 1 mM) the slower and variablemobility of the labeled products suggested poly(ADP-ribosyl)ation ofTRF1 (FIG. 9A). Inspection of Coomassie-Blue stained SDS-PAGE gels didnot reveal a larger molecular weight species upon tankyrase-mediatedTRF1 modification, indicating that only a small fraction of the TRF1 inthe reactions was modified even at high tankyrase concentrations. Thus,tankyrase functions as a processive poly(ADP-ribose) polymerase underthese conditions. TRF2 is not a substrate for modification in vitro, asmight be expected from the lack of protein-protein interactions betweenTRF2 and tankyrase.

To confirm that the labeling reaction with tankyrase was analogous toPARP-catalyzed poly(ADP-ribosyl)ation, the specific PARP inhibitor3-aminobenzamide (3AB) was added to the reactions [Purnell and Whish,Biochem J. 185:775 (1980)]. Modification of both TRF1 and tankyrase wasstrongly inhibited by 3AB (FIG. 9C). Furthermore, modified tankyrase andTRF1 reacted with a monoclonal antibody raised against poly(ADP-ribose)(FIG. 9D, see Methods above) consistent with their carrying ADP-ribosepolymers. These data indicate that tankyrase is a genuinepoly(ADP-ribose) polymerase with at least two specific substrates, TRF1and tankyrase itself.

The effect of tankyrase on the telomeric DNA binding activity of TRF1was determined by in vitro gel-shift assays using a double-strandedarray of [TTAGGG]₁₂ as a probe (see Methods, above). TRF1 binds to DNAas a homodimer and several such dimers can occupy one [TTAGGG]₁₂molecule at high TRF1 concentrations (FIG. 9E). When TRF1 was incubatedwith baculovirus-derived tankyrase in absence of NAD⁺, a slightstimulation of the TRF1 DNA binding activity occurred, resulting in theformation of higher order complexes especially at high tankyraseconcentrations. However, this stimulation of TRF1 also occurred withtotal insect cell protein, and was therefore unlikely to represent aspecific effect of tankyrase. A similar non-specific enhancement of TRF1was previously reported for β-casein and several other proteins [Chonget al. Science 270:1663 (1995)]. In contrast, when NAD⁺ was included inthe TRF1-tankyrase mixtures, a drastic reduction of the TRF1 activityresulted (FIG. 9E). This effect was dependent on the addition of activetankyrase (FIG. 9E), consistent with ADP-ribosylation being the cause ofthe TRF1 inhibition.

Discussion

TRF1 Mediates Localization of Tankyrase to Telomeres:

Only a fraction of total cellular tankyrase resided in the nucleus atthe telomere. The present data indicated that, in fact, transfectedtankyrase was excluded from the nucleus. Inspection of the primarysequence of tankyrase does not reveal a convincing match to a consensusNLS (nuclear localization signal), raising the question of how tankyrasegets into the nucleus. The demonstration that co-transfection of TRF1with tankyrase resulted in translocation of tankyrase to the nucleus,suggested the possibility of a “piggy back” mechanism. Thus,newly-synthesized TRF1 (which contains two overlapping bipartide NLSs[Chong et al., Science, 270:1663-1667 (1995)]) could bind to the ANKrepeat domain in tankyrase and carry it to telomeres. Interestingly, arecent report identified ANK repeats within several different proteinsas cis-acting NLSs [Sachdev et al., Mol. Cell Biol., 18:2524-2534(1998)]. Thus, perhaps a more general function of ANK repeat domains isto mediate interaction between a non NLS-containing ANK repeat proteinwith an NLS-containing protein, thereby allowing regulated import of theformer by the latter. In this scenario, tankyrase localization to thetelomere could be tightly regulated by TRF1 synthesis.

An alternative and not necessarily exclusive mechanism of tankyraselocalization to telomeres might occur at mitosis, when upon break downof the nuclear envelope, tankyrase would gain access to both soluble andtelomere-bound TRF1. Interesting, in co-transfected mitotic cells,tankyrase was almost exclusively found at telomeres (FIG. 3I), whereasexogeneous TRF1 was found at telomeres and throughout the cell (FIG.3F). Similarly in cotransfected interphase cells, tankyrase co-localizedwith TRF1 to telomeres, but not with overexpressed TRF1 in thenucleoplasm (FIG. 3G). These observations suggest that tankyrase has ahigher affinity for telomere-bound versus free TRF1. Thus, the telomericcomplex could serve as a high-density source of tankyrase binding sites.A high concentration of TRF1 sites may be required for efficienttankyrase binding since two-hybrid analysis (see FIG. 4B) indicates aweak affinity for tankyrase for TRF1. Recent studies demonstrate thatTRF1 can promote parallel pairing of telomeric tracts in vitro, providedthat the telomeric tracts are long and the concentration of TRF1 is high[Griffith et al., J. Mol. Biol., 278:79-88 (1998)]. Thus it was proposedthat long telomeres with sufficient TRF1 could induce intramolecularpairing resulting in a coiled higher order structure at telomeres. Sucha substrate could provide a means for long telomeres to recruittankyrase for the negative regulation of telomere length (see model inFIG. 8B and below).

Tankyrase at Nuclear Pore Complexes:

Immunogold electronmicroscopy showed that tankyrase was locatedspecifically at the tips of the fibers that emanate from the nuclearpore complex into the cytoplasm (FIG. 5C). This location is likely to bethe entry site of a multiple docking site pathway by which substratesmove through the nuclear pore complex. Indeed, only two other mammalianproteins have been localized to the tips of the cytoplasmic fibers,SUMO1-modified RanGAP1 which like tankyrase, also localizes to themitotic centrosome [Mahajan et al., Cell, 88:97-107 (1997); Matunis etal., J. Cell Biol., 135:1457-1470 (1996)] and the nucleoporin Nup358 [Wuet al., J. Biol. Chem., 270:14209 -14213 (1995); Yokoyama et al.,Nature, 376:184-188 (1995)]. SUMO1-modified RanGAP1 and Nup358 bind toeach other [Mahajan et al., Cell, 88:97-107 (1997); Matunis et al., J.Cell Biol., 135:1457-1470 (1996)] and to Ran, the Ras-like GTPase thatserves as the molecular switch for bi-directional transport through thenuclear pore [reviewed in Rush et al., Bioessays, 18:103-112 (1996)]. Inaddition, Nup358 contains short peptide repeats (a feature common to asubset of nucleoporins) which have been proposed to serve as dockingsites for import substrate-receptor complexes as they move through thenuclear pore [Radu et al., Cell, 81:215-222 (1995)]. Tankyrase might useits SAM domain or ANK repeats to bind to the fibers or to Nup358, tolocalize to nuclear pore complex fibers.

Tankyrase's localization could be significant to the port of entry fornuclear traffic. Tankyrase could play a structural role at this site and(like ankyrins) serve as a linker between the cytoplasmic fibers of thenuclear pore complex and the cytoskeleton. At this location, itsPARP-like activity could play a role in regulating nuclear transport.Alternatively, tankyrase's location at the nuclear pore complex mayserve to provide a ready pool of tankyrase waiting to be picked up byTRF1 and translocated through the nuclear pore complex to telomeres,thus allowing its localization to telomeres to be tightly controlled byTRF1.

Tankyrase at the Centrosome: Possible Relevance to Meiosis:

It has been demonstrated by indirect immunofluorescence herein thattankyrase is not an integral component of the centrosome per se butrather is located around the pericentriolar matrix where it co-localizeswith NuMA (FIG. 6I). Like Nuda, tankyrase could be associating with themicrotubules that emerge from the centrosome. NuMA exists in a complexwith cytoplasmic dynein and dynactin and appears to be required formitotic spindle pole assembly and stabilization [Merdes et al., Cell,87:447-458 (1996)]. Therefore tankyrase, may play a role in spindlefunction or stability.

This is the first report of a protein that localizes to both telomeresand centrosomes. At first glance it is difficult to imagine a connectionbetween these two structures. Normally it is not telomeres, but rather,centromeres that associate with the mitotic centrosome. Associationbetween telomeres and the centrosome does occur but it is during meiosis(FIG. 8A), not mitosis. In mammalian cells during prophase of meiosis I(in a process that may be that may be essential for the pairing andsubsequent recombination of homologous chromosomes), telomeres attach tothe nuclear envelope and gather at one pole of the nucleus to form thebouquet stage [Bass et al., J. Cell Biol., 137:5-18 (1998); Scherthan etal., J. Cell Biol., 134:1109-1125 (1996)]. Interestingly, the base ofthe bouquet is always juxtapositioned to the centrosome and earlycytological evidence indicates a connection between the centrosome andtelomeres [Dernberg et al., In Telomeres, Blackburn and Greider, eds.(Cold Spring Harbor Press), pp. 295-338 (1995)]. Tankyrase could playmultiple roles in this process (FIG. 8A). First, tankyrase could play astructural role (like ankyrins) and mediate attachment of telomeres tothe inner nuclear membrane. Second, tankyrase could act as a sink at thecentrosome to recruit telomeres to the base of the bouquet. Consistentwith a proposed role in meiosis, abundant and alternative tankyrasetranscripts in testis tissue was observed (FIG. 2A). In addition,immunoblot analysis on purified cell populations from rate testisindicated that tankyrase was highly expressed in meiotic prophase I.Although it is not yet known if TRF1 functions in meiosis, its abilityto promote parallel pairing of telomeric tracts in vitro [Griffith etal., J. Mol. Biol., 278:79-88 (1998)] would be consistent with such arole. Interestingly Taz1p, the S. pombe telomeric protein withstructural and functional similarity to TRF1, was recently found to playa critical role in prophase of meiosis I, during the horse tail stage.Here telomeres cluster at the spindle pole body (SPB, the yeastequivalent of a centrosome) and move the nucleus to facilitate alignmentof homologous chromosomes [Chikashige et al., Science, 264:270-273(1994); Chikashige et al., EMBO J., 16:193-202 (1997)]. Taz1p, isinvolved in connecting telomeres to the SPB, the horse-tail movement,and the subsequent segregation and recombination of homologouschromosomes [Cooper et al., Nature, 392:828-831 (1998); Nimmo et al.,Nature, 392:825 -828 (1998); for review see de Lange, Nature,392:753-754 (1998)].

A Role for ADP Ribosylation in Telomere Length Regulation:

The carboxy terminal domain of tankyrase displays significant homologyto the catalytic domain of PARP, a nuclear protein that in response toDNA damage catalyzes the formation of poly(ADP-ribose) onto glutamateresidues in a protein acceptor using NAD⁺ as a substrate. This homologyreflects enzymatic activity in tankyrase since all of the key aminoacids that are required for NAD⁺ binding and catalysis are conservedbetween tankyrase and PARP. Functional studies that eliminate PARPactivity, either with inhibitor, dominant negative mutants, or genedisruption, all point to a role for PARP in the maintenance of genomeintegrity. While the most likely physiological substrate of PARP is PARPitself, other in vitro recognized substrates include histones, nuclearlamins, RNA polymerase II and DNA replication enzymes including:topoisomerase I, DNA polymerase α and β, and DNA ligase II [Oei et al.,Biochemistry, 37:1465-1469 (1998); Yoshihara et al., Biochem. Biophys.Res. Commun., 128:61-67 (1985); reviewed in Althaus and Richter, Mol.Biol. Biochem. Biophys., 37:1-237 (1987)]. Although the molecularmechanism is unknown, one model suggests that PARP acts to inhibit orblock recombination at sites of DNA damage, thus allowing normal DNArepair to occur. Inhibition of recombination could be achieved throughseveral different mechanisms: PARP could modify itself, generating astructure at DNA breaks that blocks access to recombination enzymes,PARP could modify and inactivate another protein required forrecombination, or PARP could induce changes in higher order chromatinstructure by modifying histones or other chromatin-associated proteins.

Previous studies indicate that TRF1 functions as a negative regulator oftelomere length. Since TRF1 does not affect the expression oftelomerase, it may inhibit telomerase in cis, at individual chromosomeends. According to this model (see FIG. 8B), long telomeres wouldrecruit a large mass of TRF1, resulting in inhibition of telomerase. Asa result, the telomeres would shorten until the amount of TRF1 is nolonger sufficient to inhibit the elongation reaction. Thus, telomeresare proposed to be in a dynamic equilibrium between an open state inwhich telomerase is active at the termini and a close state in which theenzyme is switched off at each individual end. Recent in vitro studieshave shown that TRF1 does not affect telomerase activity even when TRF1is positioned immediately adjacent to 3′ end used for addition of TTAGGGrepeats, suggesting that the role of TRF1 in telomerase modulation ismost likely indirect.

The identification of tankyrase now suggests an alternative mechanism bywhich TRF1 may regulate telomerase-mediated telomere maintenance. Basedon the presence of the PARP domain in tankyrase, the telomeric tankyrasecould add ADP-ribose units directly to telomerase, inactivating theenzyme or otherwise limiting the elongation reaction. Alternatively,tankyrase could act indirectly, perhaps using one of the many indirecttargets proposed for the mode of action of PARP. A potential targetcould be TRF1 itself, particularly since its amino terminal domaincontains many glutamate residues. Long telomeres preferentially couldrecruit tankyrase via interaction with TRF1, resulting in a localincrease in PARP (-like) activity on long telomeres (FIG. 8B). Inagreement, the data presented herein indicate that the amount of TRF1 ontelomeres determines the abundance of tankyrase at chromosome ends. Forinstance, tankyrase at telomeres has only been detected in cells thatharbor very long TTAGGG repeat arrays (containing large amounts of TRF1)or in cells that overexpress TRF1. Thus, in the proposed model, TRF1functions as the sensor of telomere length and tankyrase relays thissignal to telomerase via ADP ribosylation.

The identification of a telomeric poly(ADP-ribose) polymerase raises thepossibility that the function of human telomeres is regulated by thistype of protein modification. Since ADP-ribosylation usually inhibitsprotein activity [Reviewed in Altheas and Richer, Mol. Biol. BiochemBeefiest 37:1 (1987); Obi et al. Biochem. 37:1465 (1998)] tankyrasecould act as a negative regulator of another factor acting at telomeres.From the in vitro studies disclosed herein, TRF1 is currently the mostobvious candidate, since it is a substrate for tankyrase in vitro andADP-ribosylation inhibits the ability of TRF1 to bind to telomeric DNA.However, the PARP activity of tankyrase could also be directed at othertelomere-associated factors, including telomerase and (ADP-ribosyl)ationcould enhance, rather than inhibit the activity of the target protein[Ruscetti et al. J. Biol. Chem. 273:14461 (1998)] PARPs have previouslybeen implicated in the cellular response to DNA damage (9). The presenceof a PARP activity at telomeres could also indicate a role for tankyrasein the protection of telomeres from inappropriate DNA damage processingactivities.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 12(2) INFORMATION FOR SEQ ID NO:1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 4134 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: double           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CGAAGATGGC GGCGTCGCGT CGCTCTCAGC ATCATCACCA CCATCATCAA CA#ACAGCTCC     60AGCCCGCCCC AGGGGCTTCA GCGCCGCCGC CGCCACCTCC TCCCCCACTC AG#CCCTGGCC    120TGGCCCCGGG GACCACCCCA GCCTCTCCCA CGGCCAGCGG CCTGGCCCCC TT#CGCCTCCC    180CGCGGCACGG CCTAGCGCTG CCGGAGGGGG ATGGCAGTCG GGATCCGCCC GA#CAGGCCCC    240GATCCCCGGA CCCGGTTGAC GGTACCAGCT GTTGCAGTAC CACCAGCACA AT#CTGTACCG    300TCGCCGCCGC TCCCGTGGTC CCAGCGGTTT CTACTTCATC TGCCGCTGGG GT#CGCTCCCA    360ACCCAGCCGG CAGTGGCAGT AACAATTCAC CGTCGTCCTC TTCTTCCCCG AC#TTCTTCCT    420CATCTTCCTC TCCATCCTCC CCTGGATCGA GCTTGGCGGA GAGCCCCGAG GC#GGCCGGAG    480TTAGCAGCAC AGCACCACTG GGGCCTGGGG CAGCAGGACC TGGGACAGGG GT#CCCAGCAG    540TGAGCGGGGC CCTACGGGAA CTGCTGGAGG CCTGTCGCAA TGGGGACGTG TC#CCGGGTAA    600AGAGGCTGGT GGACGCGGCA AACGTAAATG CAAAGGACAT GGCCGGCCGG AA#GTCTTCTC    660CCCTGCACTT CGCTGCAGGT TTTGGAAGGA AGGATGTTGT AGAACACTTA CT#ACAGATGG    720GTGCTAATGT CCACGCTCGT GATGATGGAG GTCTCATCCC GCTTCATAAT GC#CTGTTCTT    780TTGGCCATGC TGAGGTTGTG AGTCTGTTAT TGTGCCAAGG AGCTGATCCA AA#TGCCAGGG    840ATAACTGGAA CTATACACCT CTGCATGAAG CTGCTATTAA AGGGAAGATC GA#TGTGTGCA    900TTGTGCTGCT GCAGCACGGA GCTGACCCAA ACATTCGGAA CACTGATGGG AA#ATCAGCCC    960TGGACCTGGC AGATCCTTCA GCAAAAGCTG TCCTTACAGG TGAATACAAG AA#AGACGAAC   1020TCCTAGAAGC TGCTAGGAGT GGTAATGAAG AAAAACTAAT GGCTTTACTG AC#TCCTCTAA   1080ATGTGAATTG CCATGCAAGT GATGGGCGAA AGTCGACTCC TTTACATCTA GC#AGCGGGCT   1140ACAACAGAGT TCGAATAGTT CAGCTTCTTC TTCAGCATGG TGCTGATGTT CA#TGCAAAAG   1200ACAAAGGTGG ACTTGTGCCT CTTCATAATG CATGTTCATA TGGACATTAT GA#AGTCACAG   1260AACTGCTACT AAAGCATGGA GCTTGTGTTA ATGCCATGGA TCTCTGGCAG TT#TACTCCAC   1320TGCACGAGGC TGCTTCCAAG AACCGTGTAG AAGTCTGCTC TTTGTTACTT AG#CCATGGCG   1380CTGATCCTAC GTTAGTCAAC TGCCATGGCA AAAGTGCTGT GGATATGGCT CC#AACTCCGG   1440AGCTTAGGGA GAGATTGACT TATGAATTTA AAGGTCATTC TTTACTACAA GC#AGCCAGAG   1500AAGCAGACTT AGCTAAAGTT AAAAAAACAC TCGCTCTGGA AATCATTAAT TT#CAAACAAC   1560CGCAGTCTCA TGAAACAGCA CTGCACTGTG CTGTGGCCTC TCTGCATCCC AA#ACGTAAAC   1620AAGTGACAGA ATTGTTACTT AGAAAAGGAG CAAATGTTAA TGAAAAAAAT AA#AGATTTCA   1680TGACTCCCCT GCATGTTGCA GCCGAAAGAG CCCATAATGA TGTCATGGAA GT#TCTGCATA   1740AGCATGGCGC CAAGATGAAT GCACTGGACA CCCTTGGTCA GACTGCTTTG CA#TAGAGCCG   1800CCCTAGCAGG CCACCTGCAG ACCTGCCGCC TCCTGCTGAG TTACGGCTCT GA#CCCCTCCA   1860TCATCTCCTT ACAAGGCTTC ACAGCAGCAC AGATGGGCAA TGAAGCAGTG CA#GCAGATTC   1920TGAGTGAGAG TACACCTATA CGTACTTCTG ATGTTGATTA TCGACTCTTA GA#GGCATCTA   1980AAGCTGGAGA CTTGGAAACT GTGAAGCAAC TTTGCAGCTC TCAAAATGTG AA#TTGTAGAG   2040ACTTAGAGGG CCGGCATTCC ACGCCCTTAC ACTTCGCAGC AGGCTACAAC CG#CGTGTCTG   2100TTGTAGAGTA CCTGCTACAC CACGGTGCCG ATGTCCATGC CAAAGACAAG GG#TGGCTTGG   2160TGCCCCTTCA TAATGCCTGT TCATATGGAC ACTATGAGGT GGCTGAGCTT TT#AGTAAGGC   2220ATGGGGCTTC TGTCAATGTG GCGGACTTAT GGAAATTTAC CCCTCTCCAT GA#AGCAGCAG   2280CTAAAGGAAA GTATGAAATC TGCAAGCTCC TTTTAAAACA TGGAGCAGAT CC#AACTAAAA   2340AGAACAGAGA TGGAAATACA CCTTTGGATT TGGTAAAGGA AGGAGACACA GA#TATTCAGG   2400ACTTACTGAA AGGGGATGCT GCTTTGTTGG ATGCTGCCAA GAAGGGCTGC CT#GGCAAGAG   2460TGCAGAAGCT CTGTACCCCA GAGAATATCA ACTGCAGAGA CACCCAGGGC AG#AAATTCAA   2520CCCCTCTGCA CCTGGCAGCA GGCTATAATA ACCTGGAAGT AGCTGAATAT CT#TCTAGAGC   2580ATGGAGCTGA TGTTAATGCC CAGGACAAGG GTGGTTTAAT TCCTCTTCAT AA#TGCGGCAT   2640CTTATGGGCA TGTTGACATA GCGGCTTTAT TGATAAAATA CAACACGTGT GT#AAATGCAA   2700CAGATAAGTG GGCGTTTACT CCCCTCCATG AAGCAGCCCA GAAAGGAAGG AC#GCAGCTGT   2760GCGCCCTCCT CCTAGCGCAT GGTGCAGACC CCACCATGAA GAACCAGGAA GG#CCAGACGC   2820CTCTGGATCT GGCAACAGCT GACGATATCA GAGCTTTGCT GATAGATGCC AT#GCCCCCAG   2880AGGCCTTACC TACCTGTTTT AAACCTCAGG CTACTGTAGT GAGTGCCTCT CT#GATCTCAC   2940CAGCATCCAC CCCCTCCTGC CTCTCGGCTG CCAGCAGCAT AGACAACCTC AC#TGGCCCTT   3000TAGCAGAGTT GGCCGTAGGA GGAGCCTCCA ATGCAGGGGA TGGCGCCGCG GG#AACAGAAA   3060GGAAGGAAGG AGAAGTTGCT GGTCTTGACA TGAATATCAG CCAATTTCTA AA#AAGCCTTG   3120GCCTTGAACA CCTTCGGGAT ATCTTTGAAA CAGAACAGAT TACACTAGAT GT#GTTGGCTG   3180ATATGGGTCA TGAAGAGTTG AAAGAAATAG GCATCAATGC ATATGGGCAC CG#CCACAAAT   3240TAATCAAAGG AGTAGAAAGA CTCTTAGGTG GACAACAAGG CACCAATCCT TA#TTTGACTT   3300TTCACTGTGT TAATCAGGGA ACGATTTTGC TGGATCTTGC TCCAGAAGAT AA#AGAATATC   3360AGTCAGTGGA AGAAGAGATG CAAAGTACTA TTCGAGAACA CAGAGATGGT GG#TAATGCTG   3420GCGGCATCTT CAACAGATAC AATGTCATTC GAATTCAAAA AGTTGTCAAC AA#GAAGTTGA   3480GGGAGCGGTT CTGCCACCGA CAGAAGGAAG TGTCTGAGGA GAATCACAAC CA#TCACAATG   3540AGCGCATGTT GTTTCATGGT TCTCCTTTCA TTAATGCCAT TATTCATAAA GG#GTTTGATG   3600AGCGACATGC ATACATAGGA GGAATGTTTG GGGCCGGGAT TTATTTTGCT GA#AAACTCCT   3660CAAAAAGCAA CCAATATGTT TATGGAATTG GAGGAGGAAC AGGCTGCCCT AC#ACACAAGG   3720ACAGGTCATG CTATATATGT CACAGACAAA TGCTCTTCTG TAGAGTGACC CT#TGGGAAAT   3780CCTTTCTGCA GTTTAGCACC ATGAAAATGG CCCACGCGCC TCCAGGGCAC CA#CTCAGTCA   3840TTGGTAGACC GAGCGTCAAT GGGCTGGCAT ATGCTGAATA TGTCATCTAC AG#AGGAGAAC   3900AGGCATACCC AGAGTATCTT ATCACTTACC AGATCATGAA GCCAGAAGCC CC#TTCCCAGA   3960CCGCAACAGC CGCAGAGCAG AAGACCTAGT GAATGCCTGC TGGTGAAGGC CA#GATCAGAT   4020TTCAACCTGG GACTGGATTA CAGAGGATTG TTTCTAATAA CAACATCAAT AT#TCTAGAAG   4080TCCCTGACAG CCTAGAAATA AGCTGTTTGT CTTCTATAAA GCATTGCTAT AG#TG         4134 (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1327 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein   (iii) HYPOTHETICAL: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:Met Ala Ala Ser Arg Arg Ser Gln His His Hi #s His His His Gln Gln1               5    #                10   #                15Gln Leu Gln Pro Ala Pro Gly Ala Ser Ala Pr #o Pro Pro Pro Pro Pro            20       #            25       #            30Pro Pro Leu Ser Pro Gly Leu Ala Pro Gly Th #r Thr Pro Ala Ser Pro        35           #        40           #        45Thr Ala Ser Gly Leu Ala Pro Phe Ala Ser Pr #o Arg His Gly Leu Ala    50               #    55               #    60Leu Pro Glu Gly Asp Gly Ser Arg Asp Pro Pr #o Asp Arg Pro Arg Ser65                   #70                   #75                   #80Pro Asp Pro Val Asp Gly Thr Ser Cys Cys Se #r Thr Thr Ser Thr Ile                85   #                90   #                95Cys Thr Val Ala Ala Ala Pro Val Val Pro Al #a Val Ser Thr Ser Ser            100       #           105       #           110Ala Ala Gly Val Ala Pro Asn Pro Ala Gly Se #r Gly Ser Asn Asn Ser        115           #       120           #       125Pro Ser Ser Ser Ser Ser Pro Thr Ser Ser Se #r Ser Ser Ser Pro Ser    130               #   135               #   140Ser Pro Gly Ser Ser Leu Ala Glu Ser Pro Gl #u Ala Ala Gly Val Ser145                 1 #50                 1 #55                 1 #60Ser Thr Ala Pro Leu Gly Pro Gly Ala Ala Gl #y Pro Gly Thr Gly Val                165   #               170   #               175Pro Ala Val Ser Gly Ala Leu Arg Glu Leu Le #u Glu Ala Cys Arg Asn            180       #           185       #           190Gly Asp Val Ser Arg Val Lys Arg Leu Val As #p Ala Ala Asn Val Asn        195           #       200           #       205Ala Lys Asp Met Ala Gly Arg Lys Ser Ser Pr #o Leu His Phe Ala Ala    210               #   215               #   220Gly Phe Gly Arg Lys Asp Val Val Glu His Le #u Leu Gln Met Gly Ala225                 2 #30                 2 #35                 2 #40Asn Val His Ala Arg Asp Asp Gly Gly Leu Il #e Pro Leu His Asn Ala                245   #               250   #               255Cys Ser Phe Gly His Ala Glu Val Val Ser Le #u Leu Leu Cys Gln Gly            260       #           265       #           270Ala Asp Pro Asn Ala Arg Asp Asn Trp Asn Ty #r Thr Pro Leu His Glu        275           #       280           #       285Ala Ala Ile Lys Gly Lys Ile Asp Val Cys Il #e Val Leu Leu Gln His    290               #   295               #   300Gly Ala Asp Pro Asn Ile Arg Asn Thr Asp Gl #y Lys Ser Ala Leu Asp305                 3 #10                 3 #15                 3 #20Leu Ala Asp Pro Ser Ala Lys Ala Val Leu Th #r Gly Glu Tyr Lys Lys                325   #               330   #               335Asp Glu Leu Leu Glu Ala Ala Arg Ser Gly As #n Glu Glu Lys Leu Met            340       #           345       #           350Ala Leu Leu Thr Pro Leu Asn Val Asn Cys Hi #s Ala Ser Asp Gly Arg        355           #       360           #       365Lys Ser Thr Pro Leu His Leu Ala Ala Gly Ty #r Asn Arg Val Arg Ile    370               #   375               #   380Val Gln Leu Leu Leu Gln His Gly Ala Asp Va #l His Ala Lys Asp Lys385                 3 #90                 3 #95                 4 #00Gly Gly Leu Val Pro Leu His Asn Ala Cys Se #r Tyr Gly His Tyr Glu                405   #               410   #               415Val Thr Glu Leu Leu Leu Lys His Gly Ala Cy #s Val Asn Ala Met Asp            420       #           425       #           430Leu Trp Gln Phe Thr Pro Leu His Glu Ala Al #a Ser Lys Asn Arg Val        435           #       440           #       445Glu Val Cys Ser Leu Leu Leu Ser His Gly Al #a Asp Pro Thr Leu Val    450               #   455               #   460Asn Cys His Gly Lys Ser Ala Val Asp Met Al #a Pro Thr Pro Glu Leu465                 4 #70                 4 #75                 4 #80Arg Glu Arg Leu Thr Tyr Glu Phe Lys Gly Hi #s Ser Leu Leu Gln Ala                485   #               490   #               495Ala Arg Glu Ala Asp Leu Ala Lys Val Lys Ly #s Thr Leu Ala Leu Glu            500       #           505       #           510Ile Ile Asn Phe Lys Gln Pro Gln Ser His Gl #u Thr Ala Leu His Cys        515           #       520           #       525Ala Val Ala Ser Leu His Pro Lys Arg Lys Gl #n Val Thr Glu Leu Leu    530               #   535               #   540Leu Arg Lys Gly Ala Asn Val Asn Glu Lys As #n Lys Asp Phe Met Thr545                 5 #50                 5 #55                 5 #60Pro Leu His Val Ala Ala Glu Arg Ala His As #n Asp Val Met Glu Val                565   #               570   #               575Leu His Lys His Gly Ala Lys Met Asn Ala Le #u Asp Thr Leu Gly Gln            580       #           585       #           590Thr Ala Leu His Arg Ala Ala Leu Ala Gly Hi #s Leu Gln Thr Cys Arg        595           #       600           #       605Leu Leu Leu Ser Tyr Gly Ser Asp Pro Ser Il #e Ile Ser Leu Gln Gly    610               #   615               #   620Phe Thr Ala Ala Gln Met Gly Asn Glu Ala Va #l Gln Gln Ile Leu Ser625                 6 #30                 6 #35                 6 #40Glu Ser Thr Pro Ile Arg Thr Ser Asp Val As #p Tyr Arg Leu Leu Glu                645   #               650   #               655Ala Ser Lys Ala Gly Asp Leu Glu Thr Val Ly #s Gln Leu Cys Ser Ser            660       #           665       #           670Gln Asn Val Asn Cys Arg Asp Leu Glu Gly Ar #g His Ser Thr Pro Leu        675           #       680           #       685His Phe Ala Ala Gly Tyr Asn Arg Val Ser Va #l Val Glu Tyr Leu Leu    690               #   695               #   700His His Gly Ala Asp Val His Ala Lys Asp Ly #s Gly Gly Leu Val Pro705                 7 #10                 7 #15                 7 #20Leu His Asn Ala Cys Ser Tyr Gly His Tyr Gl #u Val Ala Glu Leu Leu                725   #               730   #               735Val Arg His Gly Ala Ser Val Asn Val Ala As #p Leu Trp Lys Phe Thr            740       #           745       #           750Pro Leu His Glu Ala Ala Ala Lys Gly Lys Ty #r Glu Ile Cys Lys Leu        755           #       760           #       765Leu Leu Lys His Gly Ala Asp Pro Thr Lys Ly #s Asn Arg Asp Gly Asn    770               #   775               #   780Thr Pro Leu Asp Leu Val Lys Glu Gly Asp Th #r Asp Ile Gln Asp Leu785                 7 #90                 7 #95                 8 #00Leu Lys Gly Asp Ala Ala Leu Leu Asp Ala Al #a Lys Lys Gly Cys Leu                805   #               810   #               815Ala Arg Val Gln Lys Leu Cys Thr Pro Glu As #n Ile Asn Cys Arg Asp            820       #           825       #           830Thr Gln Gly Arg Asn Ser Thr Pro Leu His Le #u Ala Ala Gly Tyr Asn        835           #       840           #       845Asn Leu Glu Val Ala Glu Tyr Leu Leu Glu Hi #s Gly Ala Asp Val Asn    850               #   855               #   860Ala Gln Asp Lys Gly Gly Leu Ile Pro Leu Hi #s Asn Ala Ala Ser Tyr865                 8 #70                 8 #75                 8 #80Gly His Val Asp Ile Ala Ala Leu Leu Ile Ly #s Tyr Asn Thr Cys Val                885   #               890   #               895Asn Ala Thr Asp Lys Trp Ala Phe Thr Pro Le #u His Glu Ala Ala Gln            900       #           905       #           910Lys Gly Arg Thr Gln Leu Cys Ala Leu Leu Le #u Ala His Gly Ala Asp        915           #       920           #       925Pro Thr Met Lys Asn Gln Glu Gly Gln Thr Pr #o Leu Asp Leu Ala Thr    930               #   935               #   940Ala Asp Asp Ile Arg Ala Leu Leu Ile Asp Al #a Met Pro Pro Glu Ala945                 9 #50                 9 #55                 9 #60Leu Pro Thr Cys Phe Lys Pro Gln Ala Thr Va #l Val Ser Ala Ser Leu                965   #               970   #               975Ile Ser Pro Ala Ser Thr Pro Ser Cys Leu Se #r Ala Ala Ser Ser Ile            980       #           985       #           990Asp Asn Leu Thr Gly Pro Leu Ala Glu Leu Al #a Val Gly Gly Ala Ser        995           #       1000           #      1005Asn Ala Gly Asp Gly Ala Ala Gly Thr Glu Ar #g Lys Glu Gly Glu Val    1010              #   1015               #  1020Ala Gly Leu Asp Met Asn Ile Ser Gln Phe Le #u Lys Ser Leu Gly Leu1025                1030 #                1035  #               1040Glu His Leu Arg Asp Ile Phe Glu Thr Glu Gl #n Ile Thr Leu Asp Val                1045  #               1050   #              1055Leu Ala Asp Met Gly His Glu Glu Leu Lys Gl #u Ile Gly Ile Asn Ala            1060      #           1065       #          1070Tyr Gly His Arg His Lys Leu Ile Lys Gly Va #l Glu Arg Leu Leu Gly        1075          #       1080           #      1085Gly Gln Gln Gly Thr Asn Pro Tyr Leu Thr Ph #e His Cys Val Asn Gln    1090              #   1095               #  1100Gly Thr Ile Leu Leu Asp Leu Ala Pro Glu As #p Lys Glu Tyr Gln Ser1105                1110 #                1115  #               1120Val Glu Glu Glu Met Gln Ser Thr Ile Arg Gl #u His Arg Asp Gly Gly                1125  #               1130   #              1135Asn Ala Gly Gly Ile Phe Asn Arg Tyr Asn Va #l Ile Arg Ile Gln Lys            1140      #           1145       #          1150Val Val Asn Lys Lys Leu Arg Glu Arg Phe Cy #s His Arg Gln Lys Glu        1155          #       1160           #      1165Val Ser Glu Glu Asn His Asn His His Asn Gl #u Arg Met Leu Phe His    1170              #   1175               #  1180Gly Ser Pro Phe Ile Asn Ala Ile Ile His Ly #s Gly Phe Asp Glu Arg1185                1190 #                1195  #               1200His Ala Tyr Ile Gly Gly Met Phe Gly Ala Gl #y Ile Tyr Phe Ala Glu                1205  #               1210   #              1215Asn Ser Ser Lys Ser Asn Gln Tyr Val Tyr Gl #y Ile Gly Gly Gly Thr            1220      #           1225       #          1230Gly Cys Pro Thr His Lys Asp Arg Ser Cys Ty #r Ile Cys His Arg Gln        1235          #       1240           #      1245Met Leu Phe Cys Arg Val Thr Leu Gly Lys Se #r Phe Leu Gln Phe Ser    1250              #   1255               #  1260Thr Met Lys Met Ala His Ala Pro Pro Gly Hi #s His Ser Val Ile Gly1265                1270 #                1275  #               1280Arg Pro Ser Val Asn Gly Leu Ala Tyr Ala Gl #u Tyr Val Ile Tyr Arg                1285  #               1290   #              1295Gly Glu Gln Ala Tyr Pro Glu Tyr Leu Ile Th #r Tyr Gln Ile Met Lys            1300      #           1305       #          1310Pro Glu Ala Pro Ser Gln Thr Ala Thr Ala Al #a Glu Gln Lys Thr        1315          #       1320           #      1325(2) INFORMATION FOR SEQ ID NO:3:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 29 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid          (A) DESCRIPTION: /desc  #= “PRIMER”    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:TTGCGGCCGC AGACGAACTC CTAGAAGCT          #                  #            29 (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid          (A) DESCRIPTION: /desc  #= “PRIMER”    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:GCGGGCCCTA TCGAATGACA TTGTATCTGT          #                  #           30 (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid          (A) DESCRIPTION: /desc  #= “PRIMER”    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:TTGCGGCCGC GGCGGCGTCG CGTCGCT           #                  #             27 (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 18 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid          (A) DESCRIPTION: /desc  #= “PRIMER”    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:TGCGGCGTCC ACCACGGT              #                   #                  #  18 (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 4491 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: double           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO     (ix) FEATURE:          (A) NAME/KEY: CDS           (B) LOCATION: 6..2027    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:CGAAG ATG GCG GCG TCG CGT CGC TCT CAG CAT #CAT CAC CAC CAT CAT         47       Met Ala Ala Ser Arg Arg Ser #Gln His His His His His His         1           #     5             #     10 CAA CAA CAG CTC CAG CCC GCC CCA GGG GCT TC#A GCG CCG CCG CCG CCA       95Gln Gln Gln Leu Gln Pro Ala Pro Gly Ala Se #r Ala Pro Pro Pro Pro 15                  # 20                  # 25                  # 30CCT CCT CCC CCA CTC AGC CCT GGC CTG GCC CC#G GGG ACC ACC CCA GCC      143Pro Pro Pro Pro Leu Ser Pro Gly Leu Ala Pr #o Gly Thr Thr Pro Ala                 35  #                 40  #                 45TCT CCC ACG GCC AGC GGC CTG GCC CCC TTC GC#C TCC CCG CGG CAC GGC      191Ser Pro Thr Ala Ser Gly Leu Ala Pro Phe Al #a Ser Pro Arg His Gly             50      #             55      #             60CTA GCG CTG CCG GAG GGG GAT GGC AGT CGG GA#T CCG CCC GAC AGG CCC      239Leu Ala Leu Pro Glu Gly Asp Gly Ser Arg As #p Pro Pro Asp Arg Pro         65          #         70          #         75CGA TCC CCG GAC CCG GTT GAC GGT ACC AGC TG#T TGC AGT ACC ACC AGC      287Arg Ser Pro Asp Pro Val Asp Gly Thr Ser Cy #s Cys Ser Thr Thr Ser     80              #     85              #     90ACA ATC TGT ACC GTC GCC GCC GCT CCC GTG GT#C CCA GCG GTT TCT ACT      335Thr Ile Cys Thr Val Ala Ala Ala Pro Val Va #l Pro Ala Val Ser Thr 95                  #100                  #105                  #110TCA TCT GCC GCT GGG GTC GCT CCC AAC CCA GC#C GGC AGT GGC AGT AAC      383Ser Ser Ala Ala Gly Val Ala Pro Asn Pro Al #a Gly Ser Gly Ser Asn                115   #               120   #               125AAT TCA CCG TCG TCC TCT TCT TCC CCG ACT TC#T TCC TCA TCT TCC TCT      431Asn Ser Pro Ser Ser Ser Ser Ser Pro Thr Se #r Ser Ser Ser Ser Ser            130       #           135       #           140CCA TCC TCC CCT GGA TCG AGC TTG GCG GAG AG#C CCC GAG GCG GCC GGA      479Pro Ser Ser Pro Gly Ser Ser Leu Ala Glu Se #r Pro Glu Ala Ala Gly        145           #       150           #       155GTT AGC AGC ACA GCA CCA CTG GGG CCT GGG GC#A GCA GGA CCT GGG ACA      527Val Ser Ser Thr Ala Pro Leu Gly Pro Gly Al #a Ala Gly Pro Gly Thr    160               #   165               #   170GGG GTC CCA GCA GTG AGC GGG GCC CTA CGG GA#A CTG CTG GAG GCC TGT      575Gly Val Pro Ala Val Ser Gly Ala Leu Arg Gl #u Leu Leu Glu Ala Cys175                 1 #80                 1 #85                 1 #90CGC AAT GGG GAC GTG TCC CGG GTA AAG AGG CT#G GTG GAC GCG GCA AAC      623Arg Asn Gly Asp Val Ser Arg Val Lys Arg Le #u Val Asp Ala Ala Asn                195   #               200   #               205GTA AAT GCA AAG GAC ATG GCC GGC CGG AAG TC#T TCT CCC CTG CAC TTC      671Val Asn Ala Lys Asp Met Ala Gly Arg Lys Se #r Ser Pro Leu His Phe            210       #           215       #           220GCT GCA GGT TTT GGA AGG AAG GAT GTT GTA GA#A CAC TTA CTA CAG ATG      719Ala Ala Gly Phe Gly Arg Lys Asp Val Val Gl #u His Leu Leu Gln Met        225           #       230           #       235GGT GCT AAT GTC CAC GCT CGT GAT GAT GGA GG#T CTC ATC CCG CTT CAT      767Gly Ala Asn Val His Ala Arg Asp Asp Gly Gl #y Leu Ile Pro Leu His    240               #   245               #   250AAT GCC TGT TCT TTT GGC CAT GCT GAG GTT GT#G AGT CTG TTA TTG TGC      815Asn Ala Cys Ser Phe Gly His Ala Glu Val Va #l Ser Leu Leu Leu Cys255                 2 #60                 2 #65                 2 #70CAA GGA GCT GAT CCA AAT GCC AGG GAT AAC TG#G AAC TAT ACA CCT CTG      863Gln Gly Ala Asp Pro Asn Ala Arg Asp Asn Tr #p Asn Tyr Thr Pro Leu                275   #               280   #               285CAT GAA GCT GCT ATT AAA GGG AAG ATC GAT GT#G TGC ATT GTG CTG CTG      911His Glu Ala Ala Ile Lys Gly Lys Ile Asp Va #l Cys Ile Val Leu Leu            290       #           295       #           300CAG CAC GGA GCT GAC CCA AAC ATT CGG AAC AC#T GAT GGG AAA TCA GCC      959Gln His Gly Ala Asp Pro Asn Ile Arg Asn Th #r Asp Gly Lys Ser Ala        305           #       310           #       315CTG GAC CTG GCA GAT CCT TCA GCA AAA GCT GT#C CTT ACA GGT GAA TAC     1007Leu Asp Leu Ala Asp Pro Ser Ala Lys Ala Va #l Leu Thr Gly Glu Tyr    320               #   325               #   330AAG AAA GAC GAA CTC CTA GAA GCT GCT AGG AG#T GGT AAT GAA GAA AAA     1055Lys Lys Asp Glu Leu Leu Glu Ala Ala Arg Se #r Gly Asn Glu Glu Lys335                 3 #40                 3 #45                 3 #50CTA ATG GCT TTA CTG ACT CCT CTA AAT GTG AA#T TGC CAT GCA AGT GAT     1103Leu Met Ala Leu Leu Thr Pro Leu Asn Val As #n Cys His Ala Ser Asp                355   #               360   #               365GGG CGA AAG TCG ACT CCT TTA CAT CTA GCA GC#G GGC TAC AAC AGA GTT     1151Gly Arg Lys Ser Thr Pro Leu His Leu Ala Al #a Gly Tyr Asn Arg Val            370       #           375       #           380CGA ATA GTT CAG CTT CTT CTT CAG CAT GGT GC#T GAT GTT CAT GCA AAA     1199Arg Ile Val Gln Leu Leu Leu Gln His Gly Al #a Asp Val His Ala Lys        385           #       390           #       395GAC AAA GGT GGA CTT GTG CCT CTT CAT AAT GC#A TGT TCA TAT GGA CAT     1247Asp Lys Gly Gly Leu Val Pro Leu His Asn Al #a Cys Ser Tyr Gly His    400               #   405               #   410TAT GAA GTC ACA GAA CTG CTA CTA AAG CAT GG#A GCT TGT GTT AAT GCC     1295Tyr Glu Val Thr Glu Leu Leu Leu Lys His Gl #y Ala Cys Val Asn Ala415                 4 #20                 4 #25                 4 #30ATG GAT CTC TGG CAG TTT ACT CCA CTG CAC GA#G GCT GCT TCC AAG AAC     1343Met Asp Leu Trp Gln Phe Thr Pro Leu His Gl #u Ala Ala Ser Lys Asn                435   #               440   #               445CGT GTA GAA GTC TGC TCT TTG TTA CTT AGC CA#T GGC GCT GAT CCT ACG     1391Arg Val Glu Val Cys Ser Leu Leu Leu Ser Hi #s Gly Ala Asp Pro Thr            450       #           455       #           460TTA GTC AAC TGC CAT GGC AAA AGT GCT GTG GA#T ATG GCT CCA ACT CCG     1439Leu Val Asn Cys His Gly Lys Ser Ala Val As #p Met Ala Pro Thr Pro        465           #       470           #       475GAG CTT AGG GAG AGA TTG ACT TAT GAA TTT AA#A GGT CAT TCT TTA CTA     1487Glu Leu Arg Glu Arg Leu Thr Tyr Glu Phe Ly #s Gly His Ser Leu Leu    480               #   485               #   490CAA GCA GCC AGA GAA GCA GAC TTA GCT AAA GT#T AAA AAA ACA CTC GCT     1535Gln Ala Ala Arg Glu Ala Asp Leu Ala Lys Va #l Lys Lys Thr Leu Ala495                 5 #00                 5 #05                 5 #10CTG GAA ATC ATT AAT TTC AAA CAA CCG CAG TC#T CAT GAA ACA GCA CTG     1583Leu Glu Ile Ile Asn Phe Lys Gln Pro Gln Se #r His Glu Thr Ala Leu                515   #               520   #               525CAC TGT GCT GTG GCC TCT CTG CAT CCC AAA CG#T AAA CAA GTG ACA GAA     1631His Cys Ala Val Ala Ser Leu His Pro Lys Ar #g Lys Gln Val Thr Glu            530       #           535       #           540TTG TTA CTT AGA AAA GGA GCA AAT GTT AAT GA#A AAA AAT AAA GAT TTC     1679Leu Leu Leu Arg Lys Gly Ala Asn Val Asn Gl #u Lys Asn Lys Asp Phe        545           #       550           #       555ATG ACT CCC CTG CAT GTT GCA GCC GAA AGA GC#C CAT AAT GAT GTC ATG     1727Met Thr Pro Leu His Val Ala Ala Glu Arg Al #a His Asn Asp Val Met    560               #   565               #   570GAA GTT CTG CAT AAG CAT GGC GCC AAG ATG AA#T GCA CTG GAC ACC CTT     1775Glu Val Leu His Lys His Gly Ala Lys Met As #n Ala Leu Asp Thr Leu575                 5 #80                 5 #85                 5 #90GGT CAG ACT GCT TTG CAT AGA GCC GCC CTA GC#A GGC CAC CTG CAG ACC     1823Gly Gln Thr Ala Leu His Arg Ala Ala Leu Al #a Gly His Leu Gln Thr                595   #               600   #               605TGC CGC CTC CTG CTG AGT TAC GGC TCT GAC CC#C TCC ATC ATC TCC TTA     1871Cys Arg Leu Leu Leu Ser Tyr Gly Ser Asp Pr #o Ser Ile Ile Ser Leu            610       #           615       #           620CAA GGC TTC ACA GCA GCA CAG ATG GGC AAT GA#A GCA GTG CAG CAG ATT     1919Gln Gly Phe Thr Ala Ala Gln Met Gly Asn Gl #u Ala Val Gln Gln Ile        625           #       630           #       635CTG AGT GTG AGT TAC GGC TCT GAC CCC TCC AT#C ATC TCC TTA CAA GGC     1967Leu Ser Val Ser Tyr Gly Ser Asp Pro Ser Il #e Ile Ser Leu Gln Gly    640               #   645               #   650TTC ACA GCA GCA CAG ATG GGC AAT GAA GCA GT#G CAG CAG ATT CTG AGT     2015Phe Thr Ala Ala Gln Met Gly Asn Glu Ala Va #l Gln Gln Ile Leu Ser655                 6 #60                 6 #65                 6 #70GGT CAT TCG TAG ATAGTGATCA TTCTACTTCA GCCTTAATGG TG#ATCTTGAG         2067 Gly His Ser  *ACGGGAAGAT TTAGAAGGAA ATCTATCCAG CATGTCTTCA CTGTCAACAT GA#AGAGTACA   2127CCTATACGTA CTTCTGATGT TGATTATCGA CTCTTAGAGG CATCTAAAGC TG#GAGACTTG   2187GAAACTGTGA AGCAACTTTG CAGCTCTCAA AATGTGAATT GTAGAGACTT AG#AGGGCCGG   2247CATTCCACGC CCTTACACTT CGCAGCAGGC TACAACAGAG TACACCTATA CG#TACTTCTG   2307ATGTTGATTA TCGACTCTTA GAGGCATCTA AAGCTGGAGA CTTGGAAACT GT#GAAGCAAC   2367TTTGCAGCTC TCAAAATGTG AATTGTAGAG ACTTAGAGGG CCGGCATTCC AC#GCCCTTAC   2427ACTTCGCAGC AGGCTACAAC CGCGTGTCTG TTGTAGAGTA CCTGCTACAC CA#CGGTGCCG   2487ATGTCCATGC CAAAGACAAG GGTGGCTTGG TGCCCCTTCA TAATGCCTGT TC#ATATGGAC   2547ACTATGAGGT GGCTGAGCTT TTAGTAAGGC ATGGGGCTTC TGTCAATGTG GC#GGACTTAT   2607GGAAATTTAC CCCTCTCCAT GAAGCAGCAG CTAAAGGAAA GTATGAAATC TG#CAAGCTCC   2667TTTTAAAACA TGGAGCAGAT CCAACTAAAA AGAACAGAGA TGGAAATACA CC#TTTGGATT   2727TGGTAAAGGA AGGAGACACA GATATTCAGG ACTTACTGAA AGGGGATGCT GC#TTTGTTGG   2787ATGCTGCCAA GAAGGGCTGC CTGGCAAGAG TGCAGAAGCT CTGTACCCCA GA#GAATATCA   2847ACTGCAGAGA CACCCAGGGC AGAAATTCAA CCCCTCTGCA CCTGGCAGCA GG#CTATAATA   2907ACCTGGAAGT AGCTGAATAT CTTCTAGAGC ATGGAGCTGA TGTTAATGCC CA#GGACAAGG   2967GTGGTTTAAT TCCTCTTCAT AATGCGGCAT CTTATGGGCA TGTTGACATA GC#GGCTTTAT   3027TGATAAAATA CAACACGTGT GTAAATGCAA CAGATAAGTG GGCGTTTACT CC#CCTCCATG   3087AAGCAGCCCA GAAAGGAAGG ACGCAGCTGT GCGCCCTCCT CCTAGCGCAT GG#TGCAGACC   3147CCACCATGAA GAACCAGGAA GGCCAGACGC CTCTGGATCT GGCAACAGCT GA#CGATATCA   3207GAGCTTTGCT GATAGATGCC ATGCCCCCAG AGGCCTTACC TACCTGTTTT AA#ACCTCAGG   3267CTACTGTAGT GAGTGCCTCT CTGATCTCAC CAGCATCCAC CCCCTCCTGC CT#CTCGGCTG   3327CCAGCAGCAT AGACAACCTC ACTGGCCCTT TAGCAGAGTT GGCCGTAGGA GG#AGCCTCCA   3387ATGCAGGGGA TGGCGCCGCG GGAACAGAAA GGAAGGAAGG AGAAGTTGCT GG#TCTTGACA   3447TGAATATCAG CCAATTTCTA AAAAGCCTTG GCCTTGAACA CCTTCGGGAT AT#CTTTGAAA   3507CAGAACAGAT TACACTAGAT GTGTTGGCTG ATATGGGTCA TGAAGAGTTG AA#AGAAATAG   3567GCATCAATGC ATATGGGCAC CGCCACAAAT TAATCAAAGG AGTAGAAAGA CT#CTTAGGTG   3627GACAACAAGG CACCAATCCT TATTTGACTT TTCACTGTGT TAATCAGGGA AC#GATTTTGC   3687TGGATCTTGC TCCAGAAGAT AAAGAATATC AGTCAGTGGA AGAAGAGATG CA#AAGTACTA   3747TTCGAGAACA CAGAGATGGT GGTAATGCTG GCGGCATCTT CAACAGATAC AA#TGTCATTC   3807GAATTCAAAA AGTTGTCAAC AAGAAGTTGA GGGAGCGGTT CTGCCACCGA CA#GAAGGAAG   3867TGTCTGAGGA GAATCACAAC CATCACAATG AGCGCATGTT GTTTCATGGT TC#TCCTTTCA   3927TTAATGCCAT TATTCATAAA GGGTTTGATG AGCGACATGC ATACATAGGA GG#AATGTTTG   3987GGGCCGGGAT TTATTTTGCT GAAAACTCCT CAAAAAGCAA CCAATATGTT TA#TGGAATTG   4047GAGGAGGAAC AGGCTGCCCT ACACACAAGG ACAGGTCATG CTATATATGT CA#CAGACAAA   4107TGCTCTTCTG TAGAGTGACC CTTGGGAAAT CCTTTCTGCA GTTTAGCACC AT#GAAAATGG   4167CCCACGCGCC TCCAGGGCAC CACTCAGTCA TTGGTAGACC GAGCGTCAAT GG#GCTGGCAT   4227ATGCTGAATA TGTCATCTAC AGAGGAGAAC AGGCATACCC AGAGTATCTT AT#CACTTACC   4287AGATCATGAA GCCAGAAGCC CCTTCCCAGA CCGCAACAGC CGCAGAGCAG AA#GACCTAGT   4347GAATGCCTGC TGGTGAAGGC CAGATCAGAT TTCAACCTGG GACTGGATTA CA#GAGGATTG   4407TTTCTAATAA CAACATCAAT ATTCTAGAAG TCCCTGACAG CCTAGAAATA AG#CTGTTTGT   4467 CTTCTATAAA GCATTGCTAT AGTG          #                   #              4491 (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH:  673 ami#no acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:Met Ala Ala Ser Arg Arg Ser Gln His His Hi #s His His His Gln Gln  1               5  #                 10  #                 15Gln Leu Gln Pro Ala Pro Gly Ala Ser Ala Pr #o Pro Pro Pro Pro Pro             20      #             25      #             30Pro Pro Leu Ser Pro Gly Leu Ala Pro Gly Th #r Thr Pro Ala Ser Pro         35          #         40          #         45Thr Ala Ser Gly Leu Ala Pro Phe Ala Ser Pr #o Arg His Gly Leu Ala     50              #     55              #     60Leu Pro Glu Gly Asp Gly Ser Arg Asp Pro Pr #o Asp Arg Pro Arg Ser 65                  # 70                  # 75                  # 80Pro Asp Pro Val Asp Gly Thr Ser Cys Cys Se #r Thr Thr Ser Thr Ile                 85  #                 90  #                 95Cys Thr Val Ala Ala Ala Pro Val Val Pro Al #a Val Ser Thr Ser Ser            100       #           105       #           110Ala Ala Gly Val Ala Pro Asn Pro Ala Gly Se #r Gly Ser Asn Asn Ser        115           #       120           #       125Pro Ser Ser Ser Ser Ser Pro Thr Ser Ser Se #r Ser Ser Ser Pro Ser    130               #   135               #   140Ser Pro Gly Ser Ser Leu Ala Glu Ser Pro Gl #u Ala Ala Gly Val Ser145                 1 #50                 1 #55                 1 #60Ser Thr Ala Pro Leu Gly Pro Gly Ala Ala Gl #y Pro Gly Thr Gly Val                165   #               170   #               175Pro Ala Val Ser Gly Ala Leu Arg Glu Leu Le #u Glu Ala Cys Arg Asn            180       #           185       #           190Gly Asp Val Ser Arg Val Lys Arg Leu Val As #p Ala Ala Asn Val Asn        195           #       200           #       205Ala Lys Asp Met Ala Gly Arg Lys Ser Ser Pr #o Leu His Phe Ala Ala    210               #   215               #   220Gly Phe Gly Arg Lys Asp Val Val Glu His Le #u Leu Gln Met Gly Ala225                 2 #30                 2 #35                 2 #40Asn Val His Ala Arg Asp Asp Gly Gly Leu Il #e Pro Leu His Asn Ala                245   #               250   #               255Cys Ser Phe Gly His Ala Glu Val Val Ser Le #u Leu Leu Cys Gln Gly            260       #           265       #           270Ala Asp Pro Asn Ala Arg Asp Asn Trp Asn Ty #r Thr Pro Leu His Glu        275           #       280           #       285Ala Ala Ile Lys Gly Lys Ile Asp Val Cys Il #e Val Leu Leu Gln His    290               #   295               #   300Gly Ala Asp Pro Asn Ile Arg Asn Thr Asp Gl #y Lys Ser Ala Leu Asp305                 3 #10                 3 #15                 3 #20Leu Ala Asp Pro Ser Ala Lys Ala Val Leu Th #r Gly Glu Tyr Lys Lys                325   #               330   #               335Asp Glu Leu Leu Glu Ala Ala Arg Ser Gly As #n Glu Glu Lys Leu Met            340       #           345       #           350Ala Leu Leu Thr Pro Leu Asn Val Asn Cys Hi #s Ala Ser Asp Gly Arg        355           #       360           #       365Lys Ser Thr Pro Leu His Leu Ala Ala Gly Ty #r Asn Arg Val Arg Ile    370               #   375               #   380Val Gln Leu Leu Leu Gln His Gly Ala Asp Va #l His Ala Lys Asp Lys385                 3 #90                 3 #95                 4 #00Gly Gly Leu Val Pro Leu His Asn Ala Cys Se #r Tyr Gly His Tyr Glu                405   #               410   #               415Val Thr Glu Leu Leu Leu Lys His Gly Ala Cy #s Val Asn Ala Met Asp            420       #           425       #           430Leu Trp Gln Phe Thr Pro Leu His Glu Ala Al #a Ser Lys Asn Arg Val        435           #       440           #       445Glu Val Cys Ser Leu Leu Leu Ser His Gly Al #a Asp Pro Thr Leu Val    450               #   455               #   460Asn Cys His Gly Lys Ser Ala Val Asp Met Al #a Pro Thr Pro Glu Leu465                 4 #70                 4 #75                 4 #80Arg Glu Arg Leu Thr Tyr Glu Phe Lys Gly Hi #s Ser Leu Leu Gln Ala                485   #               490   #               495Ala Arg Glu Ala Asp Leu Ala Lys Val Lys Ly #s Thr Leu Ala Leu Glu            500       #           505       #           510Ile Ile Asn Phe Lys Gln Pro Gln Ser His Gl #u Thr Ala Leu His Cys        515           #       520           #       525Ala Val Ala Ser Leu His Pro Lys Arg Lys Gl #n Val Thr Glu Leu Leu    530               #   535               #   540Leu Arg Lys Gly Ala Asn Val Asn Glu Lys As #n Lys Asp Phe Met Thr545                 5 #50                 5 #55                 5 #60Pro Leu His Val Ala Ala Glu Arg Ala His As #n Asp Val Met Glu Val                565   #               570   #               575Leu His Lys His Gly Ala Lys Met Asn Ala Le #u Asp Thr Leu Gly Gln            580       #           585       #           590Thr Ala Leu His Arg Ala Ala Leu Ala Gly Hi #s Leu Gln Thr Cys Arg        595           #       600           #       605Leu Leu Leu Ser Tyr Gly Ser Asp Pro Ser Il #e Ile Ser Leu Gln Gly    610               #   615               #   620Phe Thr Ala Ala Gln Met Gly Asn Glu Ala Va #l Gln Gln Ile Leu Ser625                 6 #30                 6 #35                 6 #40Val Ser Tyr Gly Ser Asp Pro Ser Ile Ile Se #r Leu Gln Gly Phe Thr                645   #               650   #               655Ala Ala Gln Met Gly Asn Glu Ala Val Gln Gl #n Ile Leu Ser Gly His            660       #           665       #           670 Ser(2) INFORMATION FOR SEQ ID NO:9:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH:  4656 am #ino acids          (B) TYPE: nucleic acid           (C) STRANDEDNESS: double          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA   (iii) HYPOTHETICAL: NO     (ix) FEATURE:           (A) NAME/KEY: CDS          (B) LOCATION: 6..2855    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:CGAAG ATG GCG GCG TCG CGT CGC TCT CAG CAT #CAT CAC CAC CAT CAT         47       Met Ala Ala Ser Arg Arg Ser #Gln His His His His His His         1           #     5             #     10 CAA CAA CAG CTC CAG CCC GCC CCA GGG GCT TC#A GCG CCG CCG CCG CCA       95Gln Gln Gln Leu Gln Pro Ala Pro Gly Ala Se #r Ala Pro Pro Pro Pro 15                  # 20                  # 25                  # 30CCT CCT CCC CCA CTC AGC CCT GGC CTG GCC CC#G GGG ACC ACC CCA GCC      143Pro Pro Pro Pro Leu Ser Pro Gly Leu Ala Pr #o Gly Thr Thr Pro Ala                 35  #                 40  #                 45TCT CCC ACG GCC AGC GGC CTG GCC CCC TTC GC#C TCC CCG CGG CAC GGC      191Ser Pro Thr Ala Ser Gly Leu Ala Pro Phe Al #a Ser Pro Arg His Gly             50      #             55      #             60CTA GCG CTG CCG GAG GGG GAT GGC AGT CGG GA#T CCG CCC GAC AGG CCC      239Leu Ala Leu Pro Glu Gly Asp Gly Ser Arg As #p Pro Pro Asp Arg Pro         65          #         70          #         75CGA TCC CCG GAC CCG GTT GAC GGT ACC AGC TG#T TGC AGT ACC ACC AGC      287Arg Ser Pro Asp Pro Val Asp Gly Thr Ser Cy #s Cys Ser Thr Thr Ser     80              #     85              #     90ACA ATC TGT ACC GTC GCC GCC GCT CCC GTG GT#C CCA GCG GTT TCT ACT      335Thr Ile Cys Thr Val Ala Ala Ala Pro Val Va #l Pro Ala Val Ser Thr 95                  #100                  #105                  #110TCA TCT GCC GCT GGG GTC GCT CCC AAC CCA GC#C GGC AGT GGC AGT AAC      383Ser Ser Ala Ala Gly Val Ala Pro Asn Pro Al #a Gly Ser Gly Ser Asn                115   #               120   #               125AAT TCA CCG TCG TCC TCT TCT TCC CCG ACT TC#T TCC TCA TCT TCC TCT      431Asn Ser Pro Ser Ser Ser Ser Ser Pro Thr Se #r Ser Ser Ser Ser Ser            130       #           135       #           140CCA TCC TCC CCT GGA TCG AGC TTG GCG GAG AG#C CCC GAG GCG GCC GGA      479Pro Ser Ser Pro Gly Ser Ser Leu Ala Glu Se #r Pro Glu Ala Ala Gly        145           #       150           #       155GTT AGC AGC ACA GCA CCA CTG GGG CCT GGG GC#A GCA GGA CCT GGG ACA      527Val Ser Ser Thr Ala Pro Leu Gly Pro Gly Al #a Ala Gly Pro Gly Thr    160               #   165               #   170GGG GTC CCA GCA GTG AGC GGG GCC CTA CGG GA#A CTG CTG GAG GCC TGT      575Gly Val Pro Ala Val Ser Gly Ala Leu Arg Gl #u Leu Leu Glu Ala Cys175                 1 #80                 1 #85                 1 #90CGC AAT GGG GAC GTG TCC CGG GTA AAG AGG CT#G GTG GAC GCG GCA AAC      623Arg Asn Gly Asp Val Ser Arg Val Lys Arg Le #u Val Asp Ala Ala Asn                195   #               200   #               205GTA AAT GCA AAG GAC ATG GCC GGC CGG AAG TC#T TCT CCC CTG CAC TTC      671Val Asn Ala Lys Asp Met Ala Gly Arg Lys Se #r Ser Pro Leu His Phe            210       #           215       #           220GCT GCA GGT TTT GGA AGG AAG GAT GTT GTA GA#A CAC TTA CTA CAG ATG      719Ala Ala Gly Phe Gly Arg Lys Asp Val Val Gl #u His Leu Leu Gln Met        225           #       230           #       235GGT GCT AAT GTC CAC GCT CGT GAT GAT GGA GG#T CTC ATC CCG CTT CAT      767Gly Ala Asn Val His Ala Arg Asp Asp Gly Gl #y Leu Ile Pro Leu His    240               #   245               #   250AAT GCC TGT TCT TTT GGC CAT GCT GAG GTT GT#G AGT CTG TTA TTG TGC      815Asn Ala Cys Ser Phe Gly His Ala Glu Val Va #l Ser Leu Leu Leu Cys255                 2 #60                 2 #65                 2 #70CAA GGA GCT GAT CCA AAT GCC AGG GAT AAC TG#G AAC TAT ACA CCT CTG      863Gln Gly Ala Asp Pro Asn Ala Arg Asp Asn Tr #p Asn Tyr Thr Pro Leu                275   #               280   #               285CAT GAA GCT GCT ATT AAA GGG AAG ATC GAT GT#G TGC ATT GTG CTG CTG      911His Glu Ala Ala Ile Lys Gly Lys Ile Asp Va #l Cys Ile Val Leu Leu            290       #           295       #           300CAG CAC GGA GCT GAC CCA AAC ATT CGG AAC AC#T GAT GGG AAA TCA GCC      959Gln His Gly Ala Asp Pro Asn Ile Arg Asn Th #r Asp Gly Lys Ser Ala        305           #       310           #       315CTG GAC CTG GCA GAT CCT TCA GCA AAA GCT GT#C CTT ACA GGT GAA TAC     1007Leu Asp Leu Ala Asp Pro Ser Ala Lys Ala Va #l Leu Thr Gly Glu Tyr    320               #   325               #   330AAG AAA GAC GAA CTC CTA GAA GCT GCT AGG AG#T GGT AAT GAA GAA AAA     1055Lys Lys Asp Glu Leu Leu Glu Ala Ala Arg Se #r Gly Asn Glu Glu Lys335                 3 #40                 3 #45                 3 #50CTA ATG GCT TTA CTG ACT CCT CTA AAT GTG AA#T TGC CAT GCA AGT GAT     1103Leu Met Ala Leu Leu Thr Pro Leu Asn Val As #n Cys His Ala Ser Asp                355   #               360   #               365GGG CGA AAG TCG ACT CCT TTA CAT CTA GCA GC#G GGC TAC AAC AGA GTT     1151Gly Arg Lys Ser Thr Pro Leu His Leu Ala Al #a Gly Tyr Asn Arg Val            370       #           375       #           380CGA ATA GTT CAG CTT CTT CTT CAG CAT GGT GC#T GAT GTT CAT GCA AAA     1199Arg Ile Val Gln Leu Leu Leu Gln His Gly Al #a Asp Val His Ala Lys        385           #       390           #       395GAC AAA GGT GGA CTT GTG CCT CTT CAT AAT GC#A TGT TCA TAT GGA CAT     1247Asp Lys Gly Gly Leu Val Pro Leu His Asn Al #a Cys Ser Tyr Gly His    400               #   405               #   410TAT GAA GTC ACA GAA CTG CTA CTA AAG CAT GG#A GCT TGT GTT AAT GCC     1295Tyr Glu Val Thr Glu Leu Leu Leu Lys His Gl #y Ala Cys Val Asn Ala415                 4 #20                 4 #25                 4 #30ATG GAT CTC TGG CAG TTT ACT CCA CTG CAC GA#G GCT GCT TCC AAG AAC     1343Met Asp Leu Trp Gln Phe Thr Pro Leu His Gl #u Ala Ala Ser Lys Asn                435   #               440   #               445CGT GTA GAA GTC TGC TCT TTG TTA CTT AGC CA#T GGC GCT GAT CCT ACG     1391Arg Val Glu Val Cys Ser Leu Leu Leu Ser Hi #s Gly Ala Asp Pro Thr            450       #           455       #           460TTA GTC AAC TGC CAT GGC AAA AGT GCT GTG GA#T ATG GCT CCA ACT CCG     1439Leu Val Asn Cys His Gly Lys Ser Ala Val As #p Met Ala Pro Thr Pro        465           #       470           #       475GAG CTT AGG GAG AGA TTG ACT TAT GAA TTT AA#A GGT CAT TCT TTA CTA     1487Glu Leu Arg Glu Arg Leu Thr Tyr Glu Phe Ly #s Gly His Ser Leu Leu    480               #   485               #   490CAA GCA GCC AGA GAA GCA GAC TTA GCT AAA GT#T AAA AAA ACA CTC GCT     1535Gln Ala Ala Arg Glu Ala Asp Leu Ala Lys Va #l Lys Lys Thr Leu Ala495                 5 #00                 5 #05                 5 #10CTG GAA ATC ATT AAT TTC AAA CAA CCG CAG TC#T CAT GAA ACA GCA CTG     1583Leu Glu Ile Ile Asn Phe Lys Gln Pro Gln Se #r His Glu Thr Ala Leu                515   #               520   #               525CAC TGT GCT GTG GCC TCT CTG CAT CCC AAA CG#T AAA CAA GTG ACA GAA     1631His Cys Ala Val Ala Ser Leu His Pro Lys Ar #g Lys Gln Val Thr Glu            530       #           535       #           540TTG TTA CTT AGA AAA GGA GCA AAT GTT AAT GA#A AAA AAT AAA GAT TTC     1679Leu Leu Leu Arg Lys Gly Ala Asn Val Asn Gl #u Lys Asn Lys Asp Phe        545           #       550           #       555ATG ACT CCC CTG CAT GTT GCA GCC GAA AGA GC#C CAT AAT GAT GTC ATG     1727Met Thr Pro Leu His Val Ala Ala Glu Arg Al #a His Asn Asp Val Met    560               #   565               #   570GAA GTT CTG CAT AAG CAT GGC GCC AAG ATG AA#T GCA CTG GAC ACC CTT     1775Glu Val Leu His Lys His Gly Ala Lys Met As #n Ala Leu Asp Thr Leu575                 5 #80                 5 #85                 5 #90GGT CAG ACT GCT TTG CAT AGA GCC GCC CTA GC#A GGC CAC CTG CAG ACC     1823Gly Gln Thr Ala Leu His Arg Ala Ala Leu Al #a Gly His Leu Gln Thr                595   #               600   #               605TGC CGC CTC CTG CTG AGT TAC GGC TCT GAC CC#C TCC ATC ATC TCC TTA     1871Cys Arg Leu Leu Leu Ser Tyr Gly Ser Asp Pr #o Ser Ile Ile Ser Leu            610       #           615       #           620CAA GGC TTC ACA GCA GCA CAG ATG GGC AAT GA#A GCA GTG CAG CAG ATT     1919Gln Gly Phe Thr Ala Ala Gln Met Gly Asn Gl #u Ala Val Gln Gln Ile        625           #       630           #       635CTG AGT GAG AGT ACA CCT ATA CGT ACT TCT GA#T GTT GAT TAT CGA CTC     1967Leu Ser Glu Ser Thr Pro Ile Arg Thr Ser As #p Val Asp Tyr Arg Leu    640               #   645               #   650TTA GAG GCA TCT AAA GCT GGA GAC TTG GAA AC#T GTG AAG CAA CTT TGC     2015Leu Glu Ala Ser Lys Ala Gly Asp Leu Glu Th #r Val Lys Gln Leu Cys655                 6 #60                 6 #65                 6 #70AGC TCT CAA AAT GTG AAT TGT AGA GAC TTA GA#G GGC CGG CAT TCC ACG     2063Ser Ser Gln Asn Val Asn Cys Arg Asp Leu Gl #u Gly Arg His Ser Thr                675   #               680   #               685CCC TTA CAC TTC GCA GCA GGC TAC AAC CGC GT#G TCT GTT GTA GAG TAC     2111Pro Leu His Phe Ala Ala Gly Tyr Asn Arg Va #l Ser Val Val Glu Tyr            690       #           695       #           700CTG CTA CAC CAC GGT GCC GAT GTC CAT GCC AA#A GAC AAG GGT GGC TTG     2159Leu Leu His His Gly Ala Asp Val His Ala Ly #s Asp Lys Gly Gly Leu        705           #       710           #       715GTG CCC CTT CAT AAT GCC TGT TCA TAT GGA CA#C TAT GAG GTG GCT GAG     2207Val Pro Leu His Asn Ala Cys Ser Tyr Gly Hi #s Tyr Glu Val Ala Glu    720               #   725               #   730CTT TTA GTA AGG CAT GGG GCT TCT GTC AAT GT#G GCG GAC TTA TGG AAA     2255Leu Leu Val Arg His Gly Ala Ser Val Asn Va #l Ala Asp Leu Trp Lys735                 7 #40                 7 #45                 7 #50TTT ACC CCT CTC CAT GAA GCA GCA GCT AAA GG#A AAG TAT GAA ATC TGC     2303Phe Thr Pro Leu His Glu Ala Ala Ala Lys Gl #y Lys Tyr Glu Ile Cys                755   #               760   #               765AAG CTC CTT TTA AAA CAT GGA GCA GAT CCA AC#T AAA AAG AAC AGA GAT     2351Lys Leu Leu Leu Lys His Gly Ala Asp Pro Th #r Lys Lys Asn Arg Asp            770       #           775       #           780GGA AAT ACA CCT TTG GAT TTG GTA AAG GAA GG#A GAC ACA GAT ATT CAG     2399Gly Asn Thr Pro Leu Asp Leu Val Lys Glu Gl #y Asp Thr Asp Ile Gln        785           #       790           #       795GAC TTA CTG AAA GGG GAT GCT GCT TTG TTG GA#T GCT GCC AAG AAG GGC     2447Asp Leu Leu Lys Gly Asp Ala Ala Leu Leu As #p Ala Ala Lys Lys Gly    800               #   805               #   810TGC CTG GCA AGA GTG CAG AAG CTC TGT ACC CC#A GAG AAT ATC AAC TGC     2495Cys Leu Ala Arg Val Gln Lys Leu Cys Thr Pr #o Glu Asn Ile Asn Cys815                 8 #20                 8 #25                 8 #30AGA GAC ACC CAG GGC AGA AAT TCA ACC CCT CT#G CAC CTG GCA GCA GGC     2543Arg Asp Thr Gln Gly Arg Asn Ser Thr Pro Le #u His Leu Ala Ala Gly                835   #               840   #               845TAT AAT AAC CTG GAA GTA GCT GAA TAT CTT CT#A GAG CAT GGA GCT GAT     2591Tyr Asn Asn Leu Glu Val Ala Glu Tyr Leu Le #u Glu His Gly Ala Asp            850       #           855       #           860GTT AAT GCC CAG GAC AAG GGT GGT TTA ATT CC#T CTT CAT AAT GCG GCA     2639Val Asn Ala Gln Asp Lys Gly Gly Leu Ile Pr #o Leu His Asn Ala Ala        865           #       870           #       875TCT TAT GGG GGC TGC CTG GCA AGA GTG CAG AA#G CTC TGT ACC CCA GAG     2687Ser Tyr Gly Gly Cys Leu Ala Arg Val Gln Ly #s Leu Cys Thr Pro Glu    880               #   885               #   890AAT ATC AAC TGC AGA GAC ACC CAG GGC AGA AA#T TCA ACC CCT CTG CAC     2735Asn Ile Asn Cys Arg Asp Thr Gln Gly Arg As #n Ser Thr Pro Leu His895                 9 #00                 9 #05                 9 #10CTG GCA GCA GGC TAT AAT AAC CTG GAA GTA GC#T GAA TAT CTT CTA GAG     2783Leu Ala Ala Gly Tyr Asn Asn Leu Glu Val Al #a Glu Tyr Leu Leu Glu                915   #               920   #               925CAT GGA GCT GAT GTT AAT GCC CAG GAC AAG GG#T GGT TTA ATT CCT CTT     2831His Gly Ala Asp Val Asn Ala Gln Asp Lys Gl #y Gly Leu Ile Pro Leu            930       #           935       #           940CAT AAT GCG GCA TCT TAT GGG TAG TAAAAGTTGG AT#TCCAAGAC CTCCTTTCCA    2885 His Asn Ala Ala Ser Tyr Gly        945           #       950GCTTGTTGTA ATGATTAAAT GAGACCATGC ATGTGGAAAT TGCATTAACT AA#TGTAAGGC   2945ATTATAAAAA TGCAAGCATG TTGACATAGC GGCTTTATTG ATAAAATACA AC#ACGTGTGT   3005AAATGCAACA GATAAGTGGG CGTTTACTCC CCTCCATGAA GCAGCCCAGA AA#GGAAGGAC   3065GCAGCTGTGC GCCCTCCTCC TAGCGCATGG TGCAGACCCC ACCATGAAGA AC#CAGGAAGG   3125CCAGACGCCT CTGGATCTGG CAACAGCTGA CGATATCAGA GCTTTGCATG TT#GACATAGC   3185GGCTTTATTG ATAAAATACA ACACGTGTGT AAATGCAACA GATAAGTGGG CG#TTTACTCC   3245CCTCCATGAA GCAGCCCAGA AAGGAAGGAC GCAGCTGTGC GCCCTCCTCC TA#GCGCATGG   3305TGCAGACCCC ACCATGAAGA ACCAGGAAGG CCAGACGCCT CTGGATCTGG CA#ACAGCTGA   3365CGATATCAGA GCTTTGCTGA TAGATGCCAT GCCCCCAGAG GCCTTACCTA CC#TGTTTTAA   3425ACCTCAGGCT ACTGTAGTGA GTGCCTCTCT GATCTCACCA GCATCCACCC CC#TCCTGCCT   3485CTCGGCTGCC AGCAGCATAG ACAACCTCAC TGGCCCTTTA GCAGAGTTGG CC#GTAGGAGG   3545AGCCTCCAAT GCAGGGGATG GCGCCGCGGG AACAGAAAGG AAGGAAGGAG AA#GTTGCTGG   3605TCTTGACATG AATATCAGCC AATTTCTAAA AAGCCTTGGC CTTGAACACC TT#CGGGATAT   3665CTTTGAAACA GAACAGATTA CACTAGATGT GTTGGCTGAT ATGGGTCATG AA#GAGTTGAA   3725AGAAATAGGC ATCAATGCAT ATGGGCACCG CCACAAATTA ATCAAAGGAG TA#GAAAGACT   3785CTTAGGTGGA CAACAAGGCA CCAATCCTTA TTTGACTTTT CACTGTGTTA AT#CAGGGAAC   3845GATTTTGCTG GATCTTGCTC CAGAAGATAA AGAATATCAG TCAGTGGAAG AA#GAGATGCA   3905AAGTACTATT CGAGAACACA GAGATGGTGG TAATGCTGGC GGCATCTTCA AC#AGATACAA   3965TGTCATTCGA ATTCAAAAAG TTGTCAACAA GAAGTTGAGG GAGCGGTTCT GC#CACCGACA   4025GAAGGAAGTG TCTGAGGAGA ATCACAACCA TCACAATGAG CGCATGTTGT TT#CATGGTTC   4085TCCTTTCATT AATGCCATTA TTCATAAAGG GTTTGATGAG CGACATGCAT AC#ATAGGAGG   4145AATGTTTGGG GCCGGGATTT ATTTTGCTGA AAACTCCTCA AAAAGCAACC AA#TATGTTTA   4205TGGAATTGGA GGAGGAACAG GCTGCCCTAC ACACAAGGAC AGGTCATGCT AT#ATATGTCA   4265CAGACAAATG CTCTTCTGTA GAGTGACCCT TGGGAAATCC TTTCTGCAGT TT#AGCACCAT   4325GAAAATGGCC CACGCGCCTC CAGGGCACCA CTCAGTCATT GGTAGACCGA GC#GTCAATGG   4385GCTGGCATAT GCTGAATATG TCATCTACAG AGGAGAACAG GCATACCCAG AG#TATCTTAT   4445CACTTACCAG ATCATGAAGC CAGAAGCCCC TTCCCAGACC GCAACAGCCG CA#GAGCAGAA   4505GACCTAGTGA ATGCCTGCTG GTGAAGGCCA GATCAGATTT CAACCTGGGA CT#GGATTACA   4565GAGGATTGTT TCTAATAACA ACATCAATAT TCTAGAAGTC CCTGACAGCC TA#GAAATAAG   4625 CTGTTTGTCT TCTATAAAGC ATTGCTATAG TG       #                   #        4657 (2) INFORMATION FOR SEQ ID NO:10:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH:  949 ami#no acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:Met Ala Ala Ser Arg Arg Ser Gln His His Hi #s His His His Gln Gln  1               5  #                 10  #                 15Gln Leu Gln Pro Ala Pro Gly Ala Ser Ala Pr #o Pro Pro Pro Pro Pro             20      #             25      #             30Pro Pro Leu Ser Pro Gly Leu Ala Pro Gly Th #r Thr Pro Ala Ser Pro         35          #         40          #         45Thr Ala Ser Gly Leu Ala Pro Phe Ala Ser Pr #o Arg His Gly Leu Ala     50              #     55              #     60Leu Pro Glu Gly Asp Gly Ser Arg Asp Pro Pr #o Asp Arg Pro Arg Ser 65                  # 70                  # 75                  # 80Pro Asp Pro Val Asp Gly Thr Ser Cys Cys Se #r Thr Thr Ser Thr Ile                 85  #                 90  #                 95Cys Thr Val Ala Ala Ala Pro Val Val Pro Al #a Val Ser Thr Ser Ser            100       #           105       #           110Ala Ala Gly Val Ala Pro Asn Pro Ala Gly Se #r Gly Ser Asn Asn Ser        115           #       120           #       125Pro Ser Ser Ser Ser Ser Pro Thr Ser Ser Se #r Ser Ser Ser Pro Ser    130               #   135               #   140Ser Pro Gly Ser Ser Leu Ala Glu Ser Pro Gl #u Ala Ala Gly Val Ser145                 1 #50                 1 #55                 1 #60Ser Thr Ala Pro Leu Gly Pro Gly Ala Ala Gl #y Pro Gly Thr Gly Val                165   #               170   #               175Pro Ala Val Ser Gly Ala Leu Arg Glu Leu Le #u Glu Ala Cys Arg Asn            180       #           185       #           190Gly Asp Val Ser Arg Val Lys Arg Leu Val As #p Ala Ala Asn Val Asn        195           #       200           #       205Ala Lys Asp Met Ala Gly Arg Lys Ser Ser Pr #o Leu His Phe Ala Ala    210               #   215               #   220Gly Phe Gly Arg Lys Asp Val Val Glu His Le #u Leu Gln Met Gly Ala225                 2 #30                 2 #35                 2 #40Asn Val His Ala Arg Asp Asp Gly Gly Leu Il #e Pro Leu His Asn Ala                245   #               250   #               255Cys Ser Phe Gly His Ala Glu Val Val Ser Le #u Leu Leu Cys Gln Gly            260       #           265       #           270Ala Asp Pro Asn Ala Arg Asp Asn Trp Asn Ty #r Thr Pro Leu His Glu        275           #       280           #       285Ala Ala Ile Lys Gly Lys Ile Asp Val Cys Il #e Val Leu Leu Gln His    290               #   295               #   300Gly Ala Asp Pro Asn Ile Arg Asn Thr Asp Gl #y Lys Ser Ala Leu Asp305                 3 #10                 3 #15                 3 #20Leu Ala Asp Pro Ser Ala Lys Ala Val Leu Th #r Gly Glu Tyr Lys Lys                325   #               330   #               335Asp Glu Leu Leu Glu Ala Ala Arg Ser Gly As #n Glu Glu Lys Leu Met            340       #           345       #           350Ala Leu Leu Thr Pro Leu Asn Val Asn Cys Hi #s Ala Ser Asp Gly Arg        355           #       360           #       365Lys Ser Thr Pro Leu His Leu Ala Ala Gly Ty #r Asn Arg Val Arg Ile    370               #   375               #   380Val Gln Leu Leu Leu Gln His Gly Ala Asp Va #l His Ala Lys Asp Lys385                 3 #90                 3 #95                 4 #00Gly Gly Leu Val Pro Leu His Asn Ala Cys Se #r Tyr Gly His Tyr Glu                405   #               410   #               415Val Thr Glu Leu Leu Leu Lys His Gly Ala Cy #s Val Asn Ala Met Asp            420       #           425       #           430Leu Trp Gln Phe Thr Pro Leu His Glu Ala Al #a Ser Lys Asn Arg Val        435           #       440           #       445Glu Val Cys Ser Leu Leu Leu Ser His Gly Al #a Asp Pro Thr Leu Val    450               #   455               #   460Asn Cys His Gly Lys Ser Ala Val Asp Met Al #a Pro Thr Pro Glu Leu465                 4 #70                 4 #75                 4 #80Arg Glu Arg Leu Thr Tyr Glu Phe Lys Gly Hi #s Ser Leu Leu Gln Ala                485   #               490   #               495Ala Arg Glu Ala Asp Leu Ala Lys Val Lys Ly #s Thr Leu Ala Leu Glu            500       #           505       #           510Ile Ile Asn Phe Lys Gln Pro Gln Ser His Gl #u Thr Ala Leu His Cys        515           #       520           #       525Ala Val Ala Ser Leu His Pro Lys Arg Lys Gl #n Val Thr Glu Leu Leu    530               #   535               #   540Leu Arg Lys Gly Ala Asn Val Asn Glu Lys As #n Lys Asp Phe Met Thr545                 5 #50                 5 #55                 5 #60Pro Leu His Val Ala Ala Glu Arg Ala His As #n Asp Val Met Glu Val                565   #               570   #               575Leu His Lys His Gly Ala Lys Met Asn Ala Le #u Asp Thr Leu Gly Gln            580       #           585       #           590Thr Ala Leu His Arg Ala Ala Leu Ala Gly Hi #s Leu Gln Thr Cys Arg        595           #       600           #       605Leu Leu Leu Ser Tyr Gly Ser Asp Pro Ser Il #e Ile Ser Leu Gln Gly    610               #   615               #   620Phe Thr Ala Ala Gln Met Gly Asn Glu Ala Va #l Gln Gln Ile Leu Ser625                 6 #30                 6 #35                 6 #40Glu Ser Thr Pro Ile Arg Thr Ser Asp Val As #p Tyr Arg Leu Leu Glu                645   #               650   #               655Ala Ser Lys Ala Gly Asp Leu Glu Thr Val Ly #s Gln Leu Cys Ser Ser            660       #           665       #           670Gln Asn Val Asn Cys Arg Asp Leu Glu Gly Ar #g His Ser Thr Pro Leu        675           #       680           #       685His Phe Ala Ala Gly Tyr Asn Arg Val Ser Va #l Val Glu Tyr Leu Leu    690               #   695               #   700His His Gly Ala Asp Val His Ala Lys Asp Ly #s Gly Gly Leu Val Pro705                 7 #10                 7 #15                 7 #20Leu His Asn Ala Cys Ser Tyr Gly His Tyr Gl #u Val Ala Glu Leu Leu                725   #               730   #               735Val Arg His Gly Ala Ser Val Asn Val Ala As #p Leu Trp Lys Phe Thr            740       #           745       #           750Pro Leu His Glu Ala Ala Ala Lys Gly Lys Ty #r Glu Ile Cys Lys Leu        755           #       760           #       765Leu Leu Lys His Gly Ala Asp Pro Thr Lys Ly #s Asn Arg Asp Gly Asn    770               #   775               #   780Thr Pro Leu Asp Leu Val Lys Glu Gly Asp Th #r Asp Ile Gln Asp Leu785                 7 #90                 7 #95                 8 #00Leu Lys Gly Asp Ala Ala Leu Leu Asp Ala Al #a Lys Lys Gly Cys Leu                805   #               810   #               815Ala Arg Val Gln Lys Leu Cys Thr Pro Glu As #n Ile Asn Cys Arg Asp            820       #           825       #           830Thr Gln Gly Arg Asn Ser Thr Pro Leu His Le #u Ala Ala Gly Tyr Asn        835           #       840           #       845Asn Leu Glu Val Ala Glu Tyr Leu Leu Glu Hi #s Gly Ala Asp Val Asn    850               #   855               #   860Ala Gln Asp Lys Gly Gly Leu Ile Pro Leu Hi #s Asn Ala Ala Ser Tyr865                 8 #70                 8 #75                 8 #80Gly Gly Cys Leu Ala Arg Val Gln Lys Leu Cy #s Thr Pro Glu Asn Ile                885   #               890   #               895Asn Cys Arg Asp Thr Gln Gly Arg Asn Ser Th #r Pro Leu His Leu Ala            900       #           905       #           910Ala Gly Tyr Asn Asn Leu Glu Val Ala Glu Ty #r Leu Leu Glu His Gly        915           #       920           #       925Ala Asp Val Asn Ala Gln Asp Lys Gly Gly Le #u Ile Pro Leu His Asn    930               #   935               #   940 Ala Ala Ser Tyr Gly945                 9 #50 (2) INFORMATION FOR SEQ ID NO:11:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH:  356 ami#no acids           (B) TYPE: nucleic acid          (C) STRANDEDNESS: double           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:TGAGTTACGG CTCTGACCCC TCCATCATCT CCTTACAAGG CTTCACAGCA GC#ACAGATGG     60GCAATGAAGC AGTGCAGCAG ATTCTGAGTG GTCATTCGTA GATAGTGATC AT#TCTACTTC    120AGCCTTAATG GTGATCTTGA GACGGGAAGA TTTAGAAGGA AATCTATCCA GC#ATGTCTTC    180ACTGTCAACA TGAAGAGTAC ACCTATACGT ACTTCTGATG TTGATTATCG AC#TCTTAGAG    240GCATCTAAAG CTGGAGACTT GGAAACTGTG AAGCAACTTT GCAGCTCTCA AA#ATGTGAAT    300TGTAGAGACT TAGAGGGCCG GCATTCCACG CCCTTACACT TCGCAGCAGG CT#ACAAC       357 (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH:  522 ami#no acids           (B) TYPE: nucleic acid          (C) STRANDEDNESS: double           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:GGCTGCCTGG CAAGAGTGCA GAAGCTCTGT ACCCCAGAGA ATATCAACTG CA#GAGACACC     60CAGGGCAGAA ATTCAACCCC TCTGCACCTG GCAGCAGGCT ATAATAACCT GG#AAGTAGCT    120GAATATCTTC TAGAGCATGG AGCTGATGTT AATGCCCAGG ACAAGGGTGG TT#TAATTCCT    180CTTCATAATG CGGCATCTTA TGGGTAGTAA AAGTTGGATT CCAAGACCTC CT#TTCCAGCT    240TGTTGTAATG ATTAAATGAG ACCATGCATG TGGAAATTGC ATTAACTAAT GT#AAGGCATT    300ATAAAAATGC AAGCATGTTG ACATAGCGGC TTTATTGATA AAATACAACA CG#TGTGTAAA    360TGCAACAGAT AAGTGGGCGT TTACTCCCCT CCATGAAGCA GCCCAGAAAG GA#AGGACGCA    420GCTGTGCGCC CTCCTCCTAG CGCATGGTGC AGACCCCACC ATGAAGAACC AG#GAAGGCCA    480 GACGCCTCTG GATCTGGCAA CAGCTGACGA TATCAGAGCT TTG    #                   #523

What is claimed is:
 1. An isolated nucleic acid encoding a vertebratetankyrase that binds to telomeric repeat binding factor 1 (TRF1),wherein the vertebrate tankyrase has an amino acid sequence that has atleast 25% identity with that of SEQ ID NO:2, and comprises: a) anankyrin-specific (ANK) repeat consensus domain; b) a sterile alpha motif(SAM), and c) a poly(ADP-ribose) polymerase (PARP)-related domain. 2.The isolated nucleic acid of claim 1 wherein the tankyrase is amammalian protein.
 3. The isolated nucleic acid of claim 2 wherein thetankyrase is a human protein.
 4. The isolated nucleic acid of claim 3wherein the tankyrase is a human protein comprising the amino acidsequence of SEQ ID NO:2, or SEQ ID NO:2 with a conservative amino acidsubstitution.
 5. The isolated nucleic acid of claim 4 wherein thenucleic acid comprises the coding sequence of SEQ ID NO:1.
 6. Theisolated nucleic acid of claim 1 further comprising a heterologousnucleotide sequence.
 7. A recombinant DNA molecule that is operativelylinked to an expression control sequence, wherein the recombinant DNAmolecule comprises the nucleic acid of claim
 1. 8. An expression vectorcontaining the recombinant DNA molecule of claim
 7. 9. A method ofexpressing a recombinant tankyrase protein in a cell containing theexpression vector of claim 8 comprising culturing the cell in anappropriate cell culture medium under conditions that provide forexpression of recombinant tankyrase by the cell.
 10. The method of claim9 further comprising the step of purifying the recombinant tankyrase.11. A recombinant DNA molecule comprising a nucleotide sequence encodinga fragment of a tankyrase that can bind to the acidic domain of a TRF1;wherein said fragment comprises at least a portion of the ANK repeatconsensus domain of the tankyrase; and wherein said tankyrase comprises:a) an ankyrin-specific (ANK) repeat consensus domain; b) a sterile alphamotif (SAM); and c) a poly(ADP-ribose) polymerase (PARP)-related domain;and wherein said tankyrase has poly(ADP-ribosyl)ating activity.
 12. Therecombinant DNA molecule of claim 11 further comprising a heterologousnucleotide sequence.
 13. The recombinant DNA molecule of claim 11wherein said fragment of the tankyrase comprises the amino acids 436 to796 of SEQ ID NO:2, or the amino acids 436 to 796 of SEQ ID NO:2 with aconservative amino acid substitution.
 14. The recombinant DNA moleculeof claim 13 wherein said fragment of the tankyrase comprises the aminoacids 181 to 1005 of SEQ ID NO:2, or the amino acids 181 to 1005 of SEQID NO:2 with a conservative amino acid substitution.
 15. The recombinantDNA molecule of claim 13 wherein said fragment of the tankyrasecomprises the amino acids 336 to 1163 of SEQ ID NO:2, or the amino acids336 to 1163 of SEQ ID NO:2 with a conservative amino acid substitution.16. A recombinant DNA molecule comprising a nucleotide sequence encodinga fragment of a tankyrase comprising the PARP domain comprising theamino acids 1159 to 1314 of SEQ ID NO:2, or the amino acids 1159 to 1314of SEQ ID NO:2 with a conservative amino acid substitution.
 17. Arecombinant DNA molecule comprising a nucleotide sequence encoding afragment of a tankyrase comprising a SAM motif comprising the aminoacids 1023 to 1088 of SEQ ID NO:2, or the amino acids 1023 to 1088 ofSEQ ID NO:2 with a conservative amino acid substitution.
 18. Arecombinant DNA molecule that is operatively linked to an expressioncontrol sequence, wherein the recombinant DNA molecule comprises thenucleic acid of claim
 11. 19. An expression vector containing therecombinant DNA molecule of claim
 18. 20. A method of expressing arecombinant tankyrase fragment in a cell containing the expressionvector of claim 19 comprising culturing the cell in an appropriate cellculture medium under conditions that provide for expression ofrecombinant tankyrase fragment by the cell.
 21. The method of claim 20further comprising the step of purifying the recombinant tankyrasefragment.